cDNA-DERIVED NUCLEIC ACIDS ENCODING RED-SHIFTED CHANNELRHODOPSINS

ABSTRACT

Methods and compositions are used to identify and characterize new channelrhodopsins derived from algae and several of which are red-shifted. The rhodopsin domain of these red-shifted channelrhodopsins can be cloned and expressed in mammalian systems and used in optogenetic applications and as therapeutic agents. Also provided are methods and compositions for use in red-shifting the absorbance maxima of channelrhodopsins in order to improve their utility for use in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This present application is a continuation of U.S. patent applicationSer. No. 13/420,352, which claims the benefit of U.S. ProvisionalApplication Ser. No. 61/452,513 filed Mar. 14, 2011, which are bothherein incorporated by reference in their entireties for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under Grant Nos.RC1AG035779 and R37GM027750 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

TECHNICAL FIELD

This disclosure generally relates to methods and compositions thatutilize channelrhodopsins derived from algae, and more particularly tosuch channelrhodopsins having red-shifted improved characteristics foroptogenetic applications or for use as therapeutic agents.

BACKGROUND

Optogenetics (reviewed by Deisseroth. Nat. Methods 8 (1): 26-9, 2011),refers to a rapidly adapted approach of using new high-speed opticalmethods for probing and controlling genetically targeted neurons withinintact neural circuits. Optogenetics involves the introduction oflight-activated channels and enzymes that allow manipulation of neuralactivity with millisecond precision while maintaining cell-typeresolution through the use of specific targeting mechanisms. Because thebrain is a high-speed system, millisecond-scale temporal precision iscentral to the concept of optogenetics, which allows probing the causalrole of specific action potential patterns in defined cells.

As traditional genetics has made use of “loss-of-function” or “gain offunction” changes that result to determine the role and expressionpattern of a particular protein. Similarly, optogenetics by definitionwill allow addition or deletion of precise activity patterns withinspecific cells in the brains of intact animals, including mammals inorder to probe the role of a particular neural function. By achievingphotonic control of neuronal firing control the action potentialpatterns involved in mammalian behavior can be determined andmanipulated.

Light control of motility behavior (phototaxis and photophobicresponses) in green flagellate algae is mediated by sensory rhodopsinshomologous to phototaxis receptors and light-driven ion transporters inprokaryotic organisms. In the phototaxis process, excitation of thealgal sensory rhodopsins leads to generation of transmembranephotoreceptor currents. When expressed in animal cells, the algalphototaxis receptors function as light-gated cation channels, which hasearned them the name “channelrhodopsins.” Channelrhodopsins have becomeuseful molecular tools for light control of cellular activity.

Originally, the source of these light-activated channels and enzymeswere several microbial opsins, including, Channelrhodopsin-2 (ChR2) asingle-component light-activated cation channel from algae, whichallowed millisecond-scale temporal control in mammals, required only onegene to be expressed in order to work, and responded to visible-spectrumlight with a chromophore (retinal) that was already present and suppliedto ChR2 by the mammalian brain tissue. The experimental utility of ChR2was quickly proven in a variety of animal models ranging from behavingmammals to classical model organisms such as flies, worms, andzebrafish, and hundreds of groups have employed ChR2 and relatedmicrobial proteins to study neural circuits.

Four channelrhodopsins have been identified to date, ChR1 and ChR2 fromChlamydomonas reinhardtii (Sineshchekov, O. A., K.-H. Jung, and J. L.Spudich. Two rhodopsins mediate phototaxis to low- and high-intensitylight in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA.99:8689-869, 2002; Nagel, G., D. Ollig, M. Fuhrmann, S. Kateriya, A. M.Musti, E. Bamberg, and P. Hegemann. Channelrhodopsin-1: a light-gatedproton channel in green algae. Science. 296:2395-2398, 2002; Nagel, G.,T. Szellas, W. Huhn, S. Kateriya, N. Adeishvili, P. Berthold, D. Ollig,P. Hegemann, and E. Bamberg. Channelrhodopsin-2, a directly light-gatedcation-selective membrane channel. Proc. Natl. Acad. Sci. USA.100:13940-13945, 2003; Suzuki, T., K. Yamasaki, S. Fujita, K. Oda, M.Iseki, K. Yoshida, M. Watanabe, H. Daiyasu, H. Toh, E. Asamizu, S.Tabata, K. Miura, H. Fukuzawa, S. Nakamura, and T. Takahashi.Archaeal-type rhodopsins in Chlamydomonas: model structure andintracellular localization. Biochem. Biophys. Res. Commun. 301:711-717,2003), and VChR1 and VChR2 from Volvox carteri (Zhang, F., M. Prigge, F.Beyriere, S. P. Tsunoda, J. Mattis, O. Yizhar, P. Hegemann, and K.Deisseroth. Red-shifted optogenetic excitation: a tool for fast neuralcontrol derived from Volvox carteri. Nat. Neurosci. 11:631-633, 2008;Kianianmomeni, A., K. Stehfest, G. Nematollahi, P. Hegemann, and A.Hallmann. Channelrhodopsins of Volvox carteri are photochromic proteinsthat are specifically expressed in somatic cells under control of light,temperature, and the sex inducer. Plant. Physiol. 151:347-366, 2009).They contain a 7-transmembrane-helix (7TM) domain characteristic of type1 rhodopsins (Spudich, J. L., C.-S. Yang, K.-H. Jung, and E. N. Spudich.Retinylidene proteins: structures and functions from archaea to humans.Annu. Rev. Cell Dev. Biol. 16:365-392, 2000) followed by a conserved butmore variable extended C-terminal region. The property of light-gatedion permeability exhibited by their 7TM domains, makes channelrhodopsinsvaluable tools for light-induced depolarization of cell membranes. Whentransfected into and expressed in excitable cells, e.g. definedsubpopulations of rodent brain neurons, channelrhodopsins enabletargeted light-activation of neuron firing in tissue culture and inliving organisms (Boyden, E. S., F. Zhang, E. Bamberg, G. Nagel, and K.Deisseroth. Millisecond-time scale, genetically targeted optical controlof neural activity. Nat. Neurosci. 8:1263-1268, 2005; Li, X., D. V.Gutierrez, M. G. Hanson, J. Han, M. D. Mark, H. Chiel, P. Hegemann, L.T. Landmesser, and S. Herlitze. Fast noninvasive activation andinhibition of neural and network activity by vertebrate rhodopsin andgreen algae channelrhodopsin. Proc. Natl. Acad. Sci. USA.102:17816-17821, 2005; Nagel, G., M. Brauner, J. F. Liewald, N.Adeishvili, E. Bamberg, and A. Gottschalk. Light activation ofchannelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggersrapid behavioral responses. Curr. Biol. 15:2279-2284, 2005). Thisoptogenetic approach, offers temporal and spatial resolution superior tothat of conventional electrical or chemical stimulation. Today,channelrhodopsins are widely used in both neuronal and non-neuronalsystems, such as glial, muscle and embryonic stem cells as a tool forcontrolled plasma membrane depolarization (reviewed by Deisseroth, 2011,ibid).

Several intrinsic properties of the four known channelrhodopsins limittheir application as optogenetic tools (reviewed in Lin, J. Y. A user'sguide to channelrhodopsin variants: features, limitations and futuredevelopments. Exp. Physiol. 96:19-25, 2010; Hegemann, P., and A.Moglich. Channelrhodopsin engineering and exploration of new optogenetictools. Nat. Methods. 8:39-42, 2011). The most widely used ChR2 hasmaximal spectral sensitivity at 470 nm but excitation at longerwavelengths is preferable to minimize light scattering by biologicaltissues. VChR1 is a red-shifted channelrhodopsin variant, but it hasslower current kinetics compromising the fidelity of neuronal spiking atmoderate to high stimulation frequencies. Another limiting property isthat photocurrents generated by all channelrhodopsins in response to apulse of continuous light decrease to a plateau level, a process called“inactivation.” In the most commonly used ChR2 this decrease can be aslarge as 80% from the peak amplitude, which correspondingly decreasesthe light-induced membrane depolarization, requiring more intense orlonger light pulses to trigger neuronal action potentials or induceother biological action. This limitation is further aggravated by lowunitary conductance of channelrhodopsins, which is less than that ofcommon ion channels, as estimated by the whole-cell current noiseanalysis (Feldbauer, K., D. Zimmermann, V. Pintschovius, J. Spitz, C.Bamann, and E. Bamberg. Channelrhodopsin-2 is a leaky proton pump. Proc.Natl. Acad. Sci. USA. 106:12317-12322, 2009; Lin, J. Y., M. Z. Lin, P.Steinbach, and R. Y. Tsien. Characterization of engineeredchannelrhodopsin variants with improved properties and kinetics.Biophys. J. 96:1803-1814, 2009).

SUMMARY

The presently disclosed methods and compositions are based, in part, onthe discovery and identification of certain novel channelrhodopsins,several of which are red-shifted, derived from algae that when clonedand expressed by mammalian cells were active for light-activation ofneuron firing. The use of these channelrhodopsins would improveoptogenetic techniques and applications and they can be used to aid indiagnosis, prevention, and/or treatment of neuronal or neurologicdisorders, such as but not limited to Parkinson's disease, as well asfor ocular disorders. Also described are methods and compositions ofred-shifting the absorbance maxima of channelrhodopsins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the photoelectric response in a suspension of nativecells of Mesostigma viride. Unilateral excitation with a 6-ns-laserpulse at 530 nm.

FIG. 2 depicts fluence-response dependence of the photoreceptor current.Excitation: broad-band 520 nm light from the photoflash.

FIG. 3 depicts the action spectrum of the photoreceptor current innative M. viride cells. Solid line, Gaussian fit of the main peak,maximum 531 nm, half-bandwidth ˜93 nm.

FIG. 4 illustrates a phylogenetic tree of the channelrhodopsin familyconstructed by the Neighbour-Joining method. In this figure, ChR1 ischannelrhodopsin 1 from C. reinhardtii; ChR2 is channelrhodopsin 2 fromC. reinhardtii; VChR1 is channelrhodopsin 1 from V. carteri; VChR2 ischannelrhodopsin 2 from V. carteri; and MChR1 is channelrhodopsin 1 fromM. viride.

FIG. 5 illustrates alignment of channelrhodopsin sequences (regions ofthe predicted helices B and C of the 7 TM domain). The conserved Gluresidues are shown in the residues 5-6 (*), the conserved His residue atthe proton donor position in BR (Asp96) is shown at residue 57 (&),other conserved residues of the retinal-binding pocket are shown in thelight shaded residues 45-47 and 50-51 (̂), and the important residuesthat are not conserved in MChR1 are indicated in the top row, residues6, 20, and 57, as V, A, A, respectively). In this figure, ChR1 ischannelrhodopsin 1 from C. reinhardtii (Seq ID NO:17); ChR2 ischannelrhodopsin 2 from C. reinhardtii (Seq ID NO:18); VChR1 ischannelrhodopsin 1 from V. carteri (Seq ID NO:19); VChR2 ischannelrhodopsin 2 from V. carteri (Seq ID NO:20); and MChR1 ischannelrhodopsin 1 from M. viride (Seq ID NO:16).

FIG. 6 illustrates photocurrents in HEK293 cells expressing the7TM-domain of MChR1 from M. viride recorded by the whole-cell patchclamp method at different holding potentials changed in 20 mV steps from−100 mV (the bottom trace) to +40 mV (the top trace).

FIG. 7 illustrates current-voltage dependencies of the peak current(squares, solid line) and the plateau level (circles, dashed line). Dataare the mean values±SEM of four successive scans.

FIG. 8 illustrates photocurrents in HEK293 cells expressing the7TM-domain of MChR1 from M. viride recorded by the whole-cell patchclamp method. Excitation: 530 nm, 2 s; relative light intensities,starting from the bottom trace: 100%, 20%, 7%, 3%, 0.5%.

FIG. 9 illustrates dependence of the initial slope (open squares, dashedline) and amplitudes of the peak and plateau currents (filled squaresand filled circles, respectively, solid lines) on the light intensity.Stimulating light 530 nm, 2 s; dark interval 30 s. The initial slope ofthe photocurrent was normalized to the peak value at the maximalintensity.

FIG. 10 illustrates low light-intensity action spectra of photoelectricresponses in HEK293 cells of MChR1 from M. viride at the external pH 9.0(solid squares), 7.4 (open circles) and 5.3 (open triangles). As thesedata were very close, a single B-spline solid line was drawn through theaverage values at each wavelength. Data for each spectrum are the meanvalues±SEM of 6 to 10 successive scans in opposite directions obtainedon 2 to 3 cells.

FIG. 11 illustrates low light-intensity action spectra of photoelectricresponses in HEK293 cells of VChR1, from Volvox carteri at the externalpH 7.4 (solid squares, solid line) and 5.3 (open circles, dashed line).As these data were very close, a single B-spline solid line was drawnthrough the average values at each wavelength. Data for each spectrumare the mean values±SEM of 6 to 10 successive scans in oppositedirections obtained on 2 to 3 cells.

FIG. 12 illustrates current kinetics of MChR1 from M. viride and VChR1from V. carteri (filled squares and open circles, respectively)expressed in HEK293 cells. Excitation: 530 nm, 2 s. Zero timecorresponds to the end of the light pulse. Data are the mean values±SEMof 6 to 10 cells for each pH value.

FIG. 13 illustrates dependence of the rate of the fast decay componenton the external pH of MChR1 from M. viride and VChR1 from V. carteri(filled squares and open circles, respectively) expressed in HEK293cells. Excitation: 530 nm, 2 s. Data are the mean values±SEM of 6 to 10cells for each pH value.

FIG. 14 illustrates photocurrents generated by the A116E and C147Amutants and wild-type MChR1 from M. viride in HEK293 cells. Traces werenormalized at the plateau level to reveal the differences in the decaykinetics. The increased level of the noise qualitatively reflects adecrease in the absolute current amplitudes. Excitation 530 nm, 0.5 s.Zero time corresponds to the end of the light pulse.

FIG. 15 illustrates photoresponses generated by MChR1 from M. viride andVChR1 from V. carteri expressed in HEK293 cells upon 25 Hz stimulationwith 530 nm light.

FIG. 16 illustrates the dependence of the amplitude modulation on thestimulus frequency for MChR1 from M. viride (filled squares) and VChR1from V. carteri (open circles). Data are the mean values±SEM of 8successive frequency changes in opposite direction.

FIG. 17 illustrates a phylogenetic trees of the 7TM domains of the sofar known channelopsins constructed by the neighbor joining methodCrChR1, channelrhodopsin 1 from C. reinhardtii; CrChR2, channelrhodopsin2 from C. reinhardtii; VcChR1, channelrhodopsin 1 from V. carteri;VcChR2, channelrhodopsin 2 from V. carteri; MvChR1, channelrhodopsin 1from M. viride; CaChR1, channelrhodopsin 1 from C. augustae; CyChR1,channelrhodopsin 1 from C. yellowstonensis; CraChR2, channelrhodopsin 2from C. raudensis; HpChR1 channelrhodopsin 1 from Haematococcuspluvialis (nucleotide Acc. No. JN596950).

FIG. 18 illustrates a phylogenetic trees of the C-terminal domains ofthe so far known channelopsins constructed by the neighbor joiningmethod. CrChR1, channelrhodopsin 1 from C. reinhardtii; CrChR2,channelrhodopsin 2 from C. reinhardtii; VcChR1, channelrhodopsin 1 fromV. carteri; VcChR2, channelrhodopsin 2 from V. carteri; MvChR1,channelrhodopsin 1 from M. viride; CaChR1, channelrhodopsin 1 from C.augustae; CyChR1, channelrhodopsin 1 from C. yellowstonensis; CraChR2,channelrhodopsin 2 from C. raudensis; HpChR1 channelrhodopsin 1 fromHaematococcus pluvialis (nucleotide Acc. No. JN596950).

FIG. 19 illustrates a partial alignment of Chlamydomonas channelopsinand bacteriorhodopsin (BR) sequences. In this figure, ChR1 ischannelrhodopsin 1 from C. reinhardtii (Seq ID NO:23); ChR2 ischannelrhodopsin 2 from C. reinhardtii (Seq ID NO:25); CaChR1,channelrhodopsin 1 from C. augustae (Seq ID NO:21); CyChR1 ischannelrhodopsin 1 from C. yellowstonensis (Seq ID NO:22); CrChR2 ischannelrhodopsin 2 from C. raudensis (Seq ID NO:24); and BR isbacteriorhodopsin (Seq ID NO:26). Black background indicates conservedidentical residues. The conserved Glu residues in the predicted secondhelix are shown in top row residues 88, 89, and 96 as well as middlerow, residues 3 and 7. Residues that form the retinal-binding pocket inbacteriorhodopsin (BR) are positioned at top row, residues 83, 87 and91; middle row, residues 29, 33, 34, 37, 62, 69, 85, and 91; and bottomrow residues 33, 37, 60 and 64. The molecular determinants thatdifferentiate ChR1/VChR1 from ChR2/VChR2 are positioned at top row atresidue 54; middle row at residue 93. Residues that are involved the inproton donor in bacteriorhodopsin (BR) are located in the middle row atresidue 40 (D in BR but H in others). The residues that are in thepositions of Glu194 and Glu204 in BR are located at bottom row,positions 42 and 52. A predicted glycosylation site for all ChRs islocated on the middle row, at positions 43-45. There are also additionalpredicted glycosylation sites for CaChR1 and CyChR1 located on the toprow, at position 2-3; for CrChR1 and CrChR2, there is also a site on thetop row at positions 28-30, and CrChR1 has another on the middle row, atpositions 49-51. Conserved residues known to be phosphorylated in ChR1or ChR2 on the top row at positions 88, 89 and 96, as well as on themiddle row at positions 3 and 7. Underlined characters show the regionsthat form transmembrane helices in bacteriorhodopsin (BR).

FIG. 20 illustrates typical kinetics of light-induced currents generatedin HEK293 cells by CrChR1 from C. raudensis (squares), CaChR1 from C.augustae (circles) and CyChR1 from C. yellowstonensis (triangles). Thecurrents in were normalized to the peak amplitude or the plateau level,respectively, and fitted with three exponential functions (solid lines).The excitation wavelength was 520 nm for CaChR1 and CyChR1, and 480 nmfor ChR1, which corresponded to their spectral maxima. Bath pH was 7.4,V_(hold) was −60 mV.

FIG. 21 illustrates typical kinetics of light-induced currents generatedin HEK293 cells by CrChR1 from C. raudensis (squares), CaChR1 from C.augustae (circles) and CyChR1 from C. yellowstonensis (triangles). Decayof the currents seen in FIG. 20 after 2-s illumination. The currentswere normalized to the peak amplitude or the plateau level,respectively, and fitted with two exponential functions (solid lines).The excitation wavelength was 520 nm for CaChR1 and CyChR1, and 480 nmfor ChR1, which corresponded to their spectral maxima. Bath pH was 7.4,V_(hold) was −60 mV.

FIG. 22 illustrates the dependence of peak (squares) and plateau(circles) amplitudes on the stimulus intensity for currents generated byCrChR1 from C. raudensis. Data points are the mean normalized values±SEM(n=3).

FIG. 23 illustrates the dependence of peak (squares) and plateau(circles) amplitudes on the stimulus intensity for currents generated byCaChR1 from C. augustae. Data points are the mean normalized values±SEM(n=5).

FIG. 24 illustrates the dependence of light inactivation (calculated asthe difference between the peak and plateau amplitudes shown in FIGS. 21and 22, relative to the peak amplitude) on the stimulus intensity forcurrents generated by CaChR1 from C. augustae (squares) and CrChR1 fromC. raudensis (circles).

FIG. 25 illustrates typical current-voltage relationships (I-V curves)for the plateau level measured at the end of a 2-s excitation lightpulse upon an increase of V_(hold) in 20 mV steps from −60 mV at thebath pH 7.4 (squares) and 5.4 (circles) in HEK293 cells transfected withCrChR1 from C. raudensis. The wavelength was 480 nm for CrChR1, whichcorresponded to its spectral maxima.

FIG. 26 illustrates typical current-voltage relationships (I-V curves)for the plateau level measured at the end of a 2-s excitation lightpulse upon an increase of V_(hold) in 20 mV steps from −60 mV at thebath pH 7.4 (squares) and 5.4 (circles) in HEK293 cells transfected withCaChR1 from C. augustae. The wavelength was 520 nm for CaChR1, whichcorresponded to its spectral maxima.

FIG. 27 illustrates typical current-voltage relationships (I-V curves)for the plateau level measured at the end of a 2-s excitation lightpulse upon an increase of V_(hold) in 20 mV steps from −60 mV at thebath pH 7.4 (squares) and 5.4 (circles) in HEK293 cells transfected withCyChR1 from C. yellowstonensis. The wavelength was 520 nm for CyChR1,which corresponded to its spectral maxima.

FIG. 28 illustrates normalized current decay traces recorded from cellstransfected with CrChR1 from C. raudensis at holding potential(V_(hold)) −60 mV. Traces at the bath pH 7.4 or 5.4 were recorded fromthe same cell. Zero time corresponds to the end of a 2-s excitationlight pulse. Excitation light was as in FIG. 24. Experimental data(dots) were fitted with two exponential functions (solid lines).

FIG. 29 illustrates normalized current decay traces recorded from cellstransfected with CaChR1 from C. augustae at holding potential (V_(hold))−60 mV. Traces at the bath pH 7.4 or 5.4 were recorded from the samecell. Zero time corresponds to the end of a 2-s excitation light pulse.Excitation light was as in FIG. 25. Experimental data (dots) were fittedwith two exponential functions (solid lines).

FIG. 30 illustrates normalized current decay traces recorded from cellstransfected with, CyChR1 from C. yellowstonensis at holding potential(V_(hold)) −60 mV. Traces at the bath pH 7.4 or 5.4 were recorded fromthe same cell. Zero time corresponds to the end of a 2-s excitationlight pulse. Excitation light was as in FIG. 26. Experimental data(dots) were fitted with two exponential functions (solid lines).

FIG. 31 illustrates the action spectra of photoelectric currentsgenerated in HEK293 cells by CaChR1 from C. augustae at the bath pH 7.4(squares), pH 5.4 (circles) or pH 9.0 (solid triangles). For comparison,the action spectrum of CrChR1 from C. reinhardtii measured at pH 7.4 isshown in panel A (open triangles, dashed line).

FIG. 32 illustrates the action spectra of photoelectric currentsgenerated in HEK293 cells by CyChR1 from C. yellowstonensis at the bathpH 7.4 (squares), pH 5.4 (circles) or pH 9.0 (solid triangles).

FIG. 33 shows a comparison of the absorption spectrum of purified CaChR1from C. augustae in detergent (line) with the action spectrum ofphotocurrents (open circles), pH 7.4.

FIG. 34 shows absorption spectra of E. coli cells expressing the protonpump AR-3 upon reconstitution with A1 or A2 retinal.

FIG. 35 illustrates photoinduced electrical signals by AR-3 expressed inE. coli and reconstituted with A1 or A2 retinal. Solid lines, currenttraces; dashed lines; transmembrane charge transfer (calculated as areaunder the current traces). Both sets of curves were normalized foreasier kinetics comparison.

FIG. 36 illustrates action spectra of charge movement by AR-3 expressedin E. coli cells (solid symbols, solid lines) or HEK293 cells (opensymbols, dashed lines) in the presence of A1 retinal (lines withsquares) or A2 retinal (lines with circles).

FIG. 37 illustrates action spectra of photocurrents generated by AR-3 attwo (dashed line) or three (solid squares, thick solid line) days afterthe addition of exogenous A2 retinal to HEK293 cells. Dotted line,action spectrum of photocurrent generated by A1-reconstituted AR-3 fromFIG. 36; thin solid line, smoothed difference between the 3 d dayspectrum and the 2nd day spectrum.

FIG. 38 illustrates action spectra of photoinduced currents generated byCrChR2 from C. reinhardtii in HEK cells upon incubation with A1 retinal(line with solid squares), or A2 retinal (line with solid circles). Thedashed line shows a deduced spectrum of pure A2-reconstituted CrChR2.

FIG. 39 illustrates (A) photoelectric currents generated by CrChR2 fromC. reinhardtii, reconstituted with A2 retinal in response tohigh-intensity stimuli (˜10²² photons×m²×s⁻¹) at 520 nm. (B) Comparisonof the current kinetics at low intensity (<10²⁰ photons×m²×s⁻¹) inresponse to 440 nm light, mostly absorbed by the A1retinal-reconstituted, and 530 nm light, mostly absorbed by the A2retinal-reconstituted pigment.

FIG. 40 illustrates action spectra of photocurrents generated by CrChR1from C. reinhardtii in HEK293 cells incubated with A2 retinal (opensquares, black line), or A1 retinal (solid line). The dashed line showsthe deduced spectrum of pure A2 retinal-reconstituted CrChR1.

FIG. 41 illustrates action spectra of photocurrents generated by CaChR1from C. augustae in HEK293 cells incubated with A2 retinal (solidsquares, solid line), or A1 retinal (dashed line).

FIG. 42 illustrates action spectra of photocurrents generated by MvChR1from M. viride in HEK293 cells incubated with A2 retinal (solid squares,solid line), or A1 retinal (dashed line), adopted from the examplesbelow.

FIG. 43 illustrates the extension of the long-wavelength spectralboundary of the proton pump AR-3 and various channelrhodopsins byincubation with A2 retinal. Bars show the spectral bands with more than1/e of maximal efficiency for the pigments reconstituted with A1(cross-hatched pattern) or A2 (hatched pattern) retinal. The absorptionspectrum of hemoglobin (oxidized+reduced) is shown for comparison.

FIG. 44 illustrates a theoretical estimation of the total number ofactinic photons absorbed over the visible range by the tested rhodopsinsat different depths of brain tissue (for more details see text). Solidsymbols and lines, pigments reconstituted with A2 retinal; open symbolsand dashed lines, pigments with A1 retinal. Squares AR-3; circles,CrChR1 from C. reinhardtii; triangles, CrChR2 from C. reinhardtii;inverted triangles, CaChR1 from C. augustae; diamonds, MvChR1 from M.viride.

FIG. 45 illustrates spectral shifts of AR-3 (solid circle) andchannelrhodopsins (solid squares) compared to the literature data forother microbial rhodopsins (small open circles) and visual pigments(small open squares) adapted from (Dartnall, H. J., and Lythgoe, J. N.(1965) The spectral clustering of visual pigments, Vision Res. 5,81-100; Tokunaga, F., and Ebrey, T. (1978) The blue membrane: the3-dehydroretinal-based artificial pigment of the purple membrane,Biochemistry 17, 1915-1922; Spudich, J. L., McCain, D. A., Nakanishi,K., Okabe, M., Shimizu, N., Rodman, H., Honig, B., and Bogomolni, R. A.(1986) Chromophore/protein interaction in bacterial sensory rhodopsinand bacteriorhodopsin, Biophys. J. 49, 479-483; Lanyi, J. K., Zimanyi,L., Nakanishi, K., Derguini, F., Okabe, M., and Honig, B. (1988)Chromophore/protein and chromophore/anion interactions in halorhodopsin,Biophys. J. 53, 185-191; Bridges, C. D. (1967) Spectroscopic propertiesof porphyropsins, Vision Res. 7, 349-369; Wald, G., Brown, P. K., andSmith, P. H. (1953) Cyanopsin, a new pigment of cone vision, Science118, 505-508; Parry, J. W., and Bowmaker, J. K. (2000) Visual pigmentreconstitution in intact goldfish retina using synthetic retinaldehydeisomers, Vision Res. 40, 2241-2247).

DESCRIPTION OF THE SEQUENCE LISTING

The Sequence Listing shows the amino acid sequences of three red-shiftedtype 1 rhodopsin domains (MChR1, SEQ ID NO: 1; CaChR1, SEQ ID NO: 2 andCyChR1, SEQ ID NO: 3) derived from the channelrhodopsin 1 of Mesostigmaviride (nucleic acid in SEQ ID NO: 5 and translated amino acid SEQ IDNO: 6); Chlamydomonas augustae (nucleic acid in SEQ ID NO: 7 andtranslated amino acid SEQ ID NO: 8) and Chlamydomonas yellowstonensis(nucleic acid in SEQ ID NOS: 9 and translated amino acid SEQ ID NO:10),respectively. Also provided is rhodopsin domain CraChR2 (CrChR2) of SEQID NO: 4, which was derived from Chlamydomonas raudensis (nucleic acidin SEQ ID NO: 11 and translated amino acid SEQ ID NO: 12)

DETAILED DESCRIPTION Definitions

In this disclosure, the use of the singular includes the plural, theword “a” or “an” means “at least one,” and the use of “or” means“and/or,” unless specifically stated otherwise. Furthermore, the use ofthe term “including,” as well as other forms, such as “includes” and“included,” is not limiting. Also, terms such as “element” or“component” encompass both elements and components comprising one unitand elements or components that comprise more than one unit unlessspecifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated herein byreference in their entirety for any purpose. In the event that one ormore of the incorporated literature and similar materials defines a termin a manner that contradicts the definition of that term in thisapplication, this application controls

As used herein, and unless otherwise indicated, the term neuron mediateddisorders for which the present methods and compositions may be usedinclude, but are not limited to, neuronal dysfunctions, disorders of thebrain, the central nervous system, the peripheral nervous system,neurological conditions, disorders of memory and leaning disorders,cardiac arrhythmias, Parkinson's disease, ocular disorders, spinal cordinjury among others.

As used herein, and unless otherwise indicated, the term oculardisorders for which the present methods and compositions may be used toimprove one or more parameters of vision include, but are not limitedto, developmental abnormalities that affect both anterior and posteriorsegments of the eye. Anterior segment disorders include, but are notlimited to, glaucoma, cataracts, corneal dystrophy, keratoconus.Posterior segment disorders include, but are not limited to, blindingdisorders caused by photoreceptor malfunction and/or death caused byretinal dystrophies and degenerations. Retinal disorders includecongenital stationary night blindness, age-related macular degeneration,congenital cone dystrophies, and a large group of retinitis pigmentosa(RP)— related disorders.

As used herein, and unless otherwise indicated, the terms “treat,”“treating,” “treatment” and “therapy” contemplate an action that occurswhile a patient is suffering from an ocular disorder that reduces theseverity of one or more symptoms or effects of an ocular disorder. Wherethe context allows, the terms “treat,” “treating,” and “treatment” alsorefers to actions taken toward ensuring that individuals at increasedrisk of a neuron mediated disorder or ocular disorders, are able toreceive appropriate surgical and/or other medical intervention prior toonset of a neuron mediated disorder or ocular disorder. As used herein,and unless otherwise indicated, the terms “prevent,” “preventing,” and“prevention” contemplate an action that occurs before a patient beginsto suffer from a neuron mediated disorder or ocular disorder, thatdelays the onset of, and/or inhibits or reduces the severity of a neuronmediated disorder or ocular disorder.

As used herein, and unless otherwise indicated, the terms “manage,”“managing,” and “management” encompass preventing, delaying, or reducingthe severity of a recurrence of an ocular disorder in a patient who hasalready suffered from such a disease, disorder or condition. The termsencompass modulating the threshold, development, and/or duration of theocular disorder or changing how a patient responds to a neuron mediateddisorder or ocular disorder.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide anytherapeutic benefit in the treatment or management of a neuron mediateddisorder or ocular disorder, or to delay or minimize one or moresymptoms associated with a neuron mediated disorder or ocular disorder.A therapeutically effective amount of a compound means an amount of thecompound, alone or in combination with one or more other therapiesand/or therapeutic agents that provide any therapeutic benefit in thetreatment or management of a neuron mediated disorder or oculardisorder. The term “therapeutically effective amount” can encompass anamount that alleviates a neuron mediated disorder or ocular disorder,improves or reduces an ocular disorder, improves overall therapy, orenhances the therapeutic efficacy of another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent ordelay the onset of a neuron mediated disorder or ocular disorder, or oneor more symptoms associated with an ocular disorder or prevent or delayits recurrence. A prophylactically effective amount of a compound meansan amount of the compound, alone or in combination with one or moreother treatment and/or prophylactic agent that provides a prophylacticbenefit in the prevention of a neuron mediated disorder or oculardisorder. The term “prophylactically effective amount” can encompass anamount that prevents a neuron mediated disorder or ocular disorder,improves overall prophylaxis, or enhances the prophylactic efficacy ofanother prophylactic agent. The “prophylactically effective amount” canbe prescribed prior to, for example, the development of a neuronmediated disorder or ocular disorder.

As used herein, “patient” or “subject” includes mammalian organismswhich are capable of suffering from an ocular disorder as describedherein, such as human and non-human mammals, for example, but notlimited to, rodents, mice, rats, non-human primates, companion animalssuch as dogs and cats as well as livestock, e.g., sheep, cow, horse,etc.

As used herein, the term “conservative substitution” generally refers toamino acid replacements that preserve the structure and functionalproperties of a protein or polypeptide. Such functionally equivalent(conservative substitution) peptide amino acid sequences include, butare not limited to, additions or substitutions of amino acid residueswithin the amino acid sequences encoded by a nucleotide sequence thatresult in a silent change, thus producing a functionally equivalent geneproduct. Conservative amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example: nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

As used herein, a “redshift” is a shift to longer wavelength. Incontrast a “blueshift” would be a shift to shorter wavelength. Theseterms apply to both light-emitting and light-absorbing objects.

Light control of motility behavior (phototaxis and photophobicresponses) in green flagellate algae is mediated by sensory rhodopsinshomologous to phototaxis receptors and light-driven ion transporters inprokaryotic organisms. In the phototaxis process, excitation of thealgal sensory rhodopsins leads to generation of transmembranephotoreceptor currents. When expressed in animal cells, the algalphototaxis receptors function as light-gated cation channels, which hasearned them the name “channelrhodopsins.” Channelrhodopsins have becomeuseful molecular tools for light control of cellular activity.

Described herein in some embodiments are compositions and methods foruse in generating and obtaining red-shifted channelrhodopsins from algaethat are superior to those currently available. Channelrhodopsins arephototaxis receptors that function as light-gated cation channels whentransfected into animal cells, are used for photoactivation of neuronfiring. Desired properties for such optogenetic uses are red-shiftedabsorption to minimize light-scattering by biological tissue, minimalinactivation in sustained light, and rapid kinetics of channel closureto allow high-frequency repetitive neuron photostimulation. Thepreviously identified homologs from Chlamydomonas reinhardtii and Volvoxcarteri and their genetically engineered variants exhibit some of theseproperties, but none exhibits all.

As used herein, the channelopsin is the apoprotein, whilechannelrhodopsin is the protein and retinal. Strictly speaking the aminoacid sequences (identified in SEQ ID NOS: 1-4) define the opsin, butthis is also the sequence of the rhodopsin, which is the same.

By screening phototaxis receptor currents among several algal species,several red-shifted channelrhodopsins with rapid kinetics wereidentified and characterized. In some embodiments, the disclosed methodsprovide a technology that facilities the identification andcharacterization of particularly useful channelrhodopsins from algae(such as but not limited to, Mesostigma viride, Chlamydomonas augustae,Chlamydomonas yellowstonensis, Chlorophyceae, Chlamydomonas reinhardtii,Acetabularia, Ulva, Pyramimonas, Platymonas (Tetraselmis).

In some embodiments, provided are amino acid and nucleic acid sequencesof functional domains of novel channelrhodopsins that are alsofunctionally characterized. Three of these channelrhodopsins have beendetermined to have red-shifted absorption maxima and functional type 1rhodopsin domains of the channelrhodopsins were cloned and identified asMvChR1 or MChR1 (SEQ ID NO: 1) which was derived from channelrhodopsin 1of Mesostigma viride (SEQ ID NOS: 5 and 6, EMBL entry JF922293.1);CaChR1 (SEQ ID NO: 2) which was derived from channelrhodopsin 1 ofChlamydomonas augustae (SEQ ID NO: 7 and 8, EMBL entry JN596951.1; andCyChR1 (SEQ ID NO: 3) which was derived from channelrhodopsin 1Chlamydomonas yellowstonensis (SEQ ID NOS: 9 and 10, see EMBL entryJN596948.1).

Also provided, in some embodiments, is the use and composition of afourth novel channelrhodopsin domain, identified as CraChR or CrChR (SEQID NO: 4) which was derived from channelrhodopsin 2 of Chlamydomonasraudensis (SEQ ID NOS: 11 and 12, EMBL entry JN596949.1., in onepublication this was referred to as CraChR2 (Hou, S.-Y., Govorunova, E.G., Ntefidou, M., Lane, C. E., Spudich, E. N., Sineshchekov, O. A., andSpudich, J. L. Diversity of Chlamydomonas channelrhodopsins, Photochem.Photobiol., 88: 119-128, 2012).

In other embodiments, are methods compositions that providechannelrhodopsins with improved properties and characteristics thatenhance the application of these compositions in, among other things,optogenetic techniques. These embodiments involve, among others, theenhancement of the long-wavelength sensitivity, by inducing a redshiftin absorption maxima.

Optogenetic techniques involve the introduction of light-activatedchannels and enzymes that allow manipulation of neural activity andcontrol of neuronal function. Thus, in some embodiments, the disclosedmethods and compositions can be introduced into cells and facilitate themanipulation of the cells activity and function.

Optogenetic techniques, and thus the disclosed methods and compositions,can be used to, among other things, characterize the functions ofcomplex neural circuits and information processing in the normal brainand during various neurological conditions; functionally map thecerebral cortex; characterize and manipulate the process of learning andmemory; characterize and manipulate the process of synaptic transmissionand plasticity; provide light-controlled induction of gene expression;provide optical control of cell motility and other activities.

Clinical applications of the disclosed methods and compositions include,but are not limited to, optogenetic approaches to therapy such as, butare not limited to, restoration of vision by introduction ofchannelrhodopsins in post-receptor neurons in the retina for oculardisorder gene-therapy treatment of age-dependent macular degeneration,diabetic retinopathy, and retinitis pigmentosa, as well as otherconditions which result in loss of photoreceptor cells; control ofcardiac function by using channelrhodopsins incorporated into excitablecardiac muscle cells in the atrioventricular bundle (bundle of His) tocontrol heart beat rhythm rather than an electrical pacemaker device;restoration of dopamine-related movement dysfunction in Parkinsonianpatients; amelioration of depression; recovery of breathing after spinalcord injury; provide noninvasive control of stem cell differentiationand assess specific contributions of transplanted cells to tissue andnetwork function.

In some embodiments, particular red-shifted channelrhodopsins with rapidkinetics are provided, including, MChR1 (SEQ ID NO: 1) which was derivedfrom Mesostigma viride; CaChR1 (SEQ ID NO: 2) which was derived fromChlamydomonas augustae; and CyChR1 (SEQ ID NO: 3) which was derived fromChlamydomonas yellowstonensis may be used to enhance, among otherthings, optogenetic techniques and optogenetic approaches to therapy.

Channelrhodopsins, functional or active portions thereof, such as butnot limited to the type 1 rhodopsin domain, and functional equivalentsinclude, but are not limited to, naturally occurring versions ofchannelrhodopsin and those that are orthologs and homologs, and mutantversions of channelrhodopsin, whether naturally occurring or engineered(by site directed mutagenesis, gene shuffling, or directed evolution, asdescribed in, for example, U.S. Pat. No. 5,837,458). Also included arethe use of degenerate nucleic acid variants (due to the redundancy ofthe genetic code) of the disclosed an algae derived channelrhodopsinpolynucleotide sequences.

In some embodiments, are isolated nucleic acid molecules comprising anucleotide sequence that encodes the type 1 rhodopsin domain of ared-shifted channelrhodopsin derived from algae. In some embodiments,the type 1 rhodopsin domain encodes the peptides whose sequence isdescribed in a group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ IDNO: 3. In some embodiments, are isolated nucleic acid moleculescomprising a nucleotide sequence that encodes the amino acid sequenceshown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, are expression vectors comprising a nucleic acid sequencethat encodes the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 orSEQ ID NO: 3. In some embodiments, are host cells comprising aexpression vector comprising a nucleic acid sequence that encodes theamino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In some embodiments, are isolated nucleic acid molecules comprising anucleotide sequence that encodes the rhodopsin domain of achannelrhodopsin derived from algae. In some embodiments, the rhodopsindomain encodes the peptides whose sequence is described in SEQ ID NO: 4.In some embodiments, are isolated nucleic acid molecules comprising anucleotide sequence that encodes the amino acid sequence shown in SEQ IDNO: 4. In some embodiments, are expression vectors comprising a nucleicacid sequence that encodes the amino acid sequences of SEQ ID NO: 4. Insome embodiments, are host cells comprising a expression vectorcomprising a nucleic acid sequence that encodes the amino acid sequencesof SEQ ID NO: 4.

In some embodiments, are isolated peptides comprising a sequence thatencodes the type 1 rhodopsin domain of a red-shifted channelrhodopsinderived from algae. In some embodiments, are isolated peptidescomprising an amino acid sequence of a group consisting of SEQ ID NO: 1,SEQ ID NO: 2 or SEQ ID NO: 3.

In some embodiments, are isolated peptides comprising a sequence thatencodes the rhodopsin domain of a channelrhodopsin derived from algae.In some embodiments, are isolated peptides comprising an amino acidsequence of SEQ ID NO: 4. In some embodiments, are isolated peptidescomprising an amino acid sequence of a group consisting of SEQ ID NO: 1,SEQ ID NO: 2 or SEQ ID NO: 3 and function as a channelrhodopsin. In someembodiments, are isolated peptides comprising an amino acid sequence ofSEQ ID NO: 4 and function as a channelrhodopsin.

In some embodiments, are isolated nucleic acid molecules comprising anucleotide sequence that encodes the type 1 rhodopsin of a red-shiftedchannelrhodopsin derived from algae. In some embodiments, the type 1rhodopsin encodes a peptide whose sequence is described in a groupconsisting of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. In someembodiments, are isolated nucleic acid molecules comprising a nucleotidesequence that encodes the amino acid sequence shown in SEQ ID NO: 6, SEQID NO: 8 or SEQ ID NO: 10. In some embodiments, are expression vectorscomprising a nucleic acid sequence that encodes the amino acid sequencesof SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. In some embodiments, arehost cells comprising a expression vector comprising a nucleic acidsequence that encodes the amino acid sequences of SEQ ID NO: 6, SEQ IDNO: 8 or SEQ ID NO: 10.

In some embodiments, are peptides comprising a sequence that encodes thetype 1 rhodopsin domain of a red-shifted channelrhodopsin derived fromalgae. In some embodiments, are isolated peptides comprising an aminoacid sequence of a group consisting of SEQ ID NO: 6, SEQ ID NO: 8 or SEQID NO: 10. In some embodiments, are isolated peptides comprising asequence that encodes the rhodopsin domain of channelrhodopsin derivedfrom algae. In some embodiments, the isolated peptides comprise an aminoacid sequence of SEQ ID NO: 12.

In some embodiments, are isolated nucleic acid molecules wherein saidnucleic acid molecule has a sequence is selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.In some embodiments, are expression vectors comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9 or SEQ ID NO: 11. In some embodiments, are host cellscomprising a expression vector comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9 or SEQ ID NO: 11. In some embodiments, an isolated nucleic acidcomprises a nucleotide sequence that encodes the type 1 rhodopsin domainof a red-shifted channelrhodopsin derived from algae. In someembodiments, the nucleotide sequence encodes at least 16 contiguousamino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In someembodiments, the nucleotide sequence encodes at least 20 contiguousamino acids of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, the nucleotide sequence encodes at least 35 contiguousamino acids of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, the nucleotide sequence encodes at least 50 contiguousamino acids of SEQ ID NO:1, SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, the nucleotide sequence encodes at least 75 contiguousamino acids of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In someembodiments, the nucleotide sequence encodes at least 33 contiguousamino acids of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, the nucleotide sequence encodes a peptide comprising SEQ IDNO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In some embodiments, an isolated polypeptide encodes the type 1rhodopsin domain of a red-shifted channelrhodopsin derived from algae.In some embodiments, an isolated polypeptide comprising the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In someembodiments, the isolated polypeptide has at least 85% homology to theamino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. In someembodiments, a protein composition comprises a polypeptide having theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

In some embodiments, an isolated nucleic acid comprises a nucleotidesequence that encodes the rhodopsin domain of a novel channelrhodopsinderived from C. raudensis. In some embodiments, the nucleotide sequenceencodes at least 16 contiguous amino acids of SEQ ID NO: 4. In someembodiments, the nucleotide sequence encodes at least 20 contiguousamino acids of SEQ ID NO: 4. In some embodiments, the nucleotidesequence encodes at least 35 contiguous amino acids of SEQ ID NO: 4. Insome embodiments, the nucleotide sequence encodes at least 50 contiguousamino acids of SEQ ID NO: 4. In some embodiments, the nucleotidesequence encodes at least 75 contiguous amino acids of SEQ ID NO: 4. Insome embodiments, the nucleotide sequence encodes at least 33 contiguousamino acids of SEQ ID NO: 4. In some embodiments, the nucleotidesequence encodes a peptide comprising SEQ ID NO: 4.

In some embodiments, an isolated polypeptide encodes the rhodopsindomain of a channelrhodopsin derived from C. raudensis. In someembodiments, an isolated polypeptide comprising the amino acid sequenceof SEQ ID NO: 4. In some embodiments, the isolated polypeptide has atleast 85% homology to the amino acid sequence of SEQ ID NO: 4. In someembodiments, a protein composition comprises a polypeptide having theamino acid sequence of SEQ ID NO: 4.

In some embodiments, an isolated nucleic acid comprising a nucleotidesequence that encodes a functional domain of a channelrhodopsin ofMesostigma viride, Chlamydomonas augustae, Chlamydomonas yellowstonensisor Chlamydomonas raudensis. In some embodiments are isolated nucleicacid that encodes at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 500, 600,700, 800, 900 or more contiguous amino acids of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12 or fragments thereof. Further, in some embodiments,any range derivable between any of the above-described integers.

In other embodiments, the present invention provides for an isolatedpolypeptide or an isolated nucleic acid encoding a polypeptide havingbetween about 70% and about 75%; or more preferably between about 75%and about 80%; or more preferably between about 80% and 90%; or evenmore preferably between about 90% and about 99% of amino acids that areidentical to the amino acids of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12or fragments thereof. The percent identity or homology is determinedwith regard to the length of the relevant amino acid sequence.Therefore, if a polypeptide of the present invention is comprised withina larger polypeptide, the percent homology is determined with regardonly to the portion of the polypeptide that corresponds to thepolypeptide of the present invention and not the percent homology of theentirety of the larger polypeptide.

In other embodiments, the present invention provides for an isolatednucleic acid encoding a polypeptide having between about 70% and about75%; or more preferably between about 75% and about 80%; or morepreferably between about 80% and 90%; or even more preferably betweenabout 90% and about 99% of amino acids that are identical to the aminoacids of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 orfragments thereof.

In some embodiments, the nucleic acid segments, regardless of the lengthof the coding sequence itself, may be combined with other DNA sequences,such as promoters, enhancers, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like.

In certain embodiments the invention provides an isolated nucleic acidobtained by amplification from a template nucleic acid using a primerselected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14; andSEQ ID NO: 15.

In some embodiments, a recombinant host cell comprising one of thenucleic acid sequences described. In some embodiments, a proteincomposition comprising one of the polypeptides described.

In some embodiments, are methods of treating a neuronal disorder,comprising: (a) delivering to a target neuron a nucleic acid expressionvector that encodes a type 1 rhodopsin domain of a red-shiftedchannelrhodopsin derived from algae, expressible in said target neuron,said vector comprising an open reading frame encoding the type 1rhodopsin domain of a red-shifted channelrhodopsin, operatively linkedto a promoter sequence, and optionally, a transcriptional regulatorysequence; and (b) expressing said vector in said target neuron, whereinthe expressed rhodopsin activates said target neuron upon exposure tolight. In some embodiments, the type 1 rhodopsin domain is encoded bySEQ ID NOS: 1-3.

In some embodiments, are methods of treating a neuronal disorder,comprising: (a) delivering to a target neuron a nucleic acid expressionvector that encodes a rhodopsin domain of a channelrhodopsin derivedfrom algae, expressible in said target neuron, said vector comprising anopen reading frame encoding the rhodopsin domain of a channelrhodopsin,operatively linked to a promoter sequence, and optionally, atranscriptional regulatory sequence; and (b) expressing said vector insaid target neuron, wherein the expressed rhodopsin activates saidtarget neuron upon exposure to light. In some embodiments, the rhodopsindomain is encoded by SEQ ID NO: 4.

In some embodiments, are methods of restoring light sensitivity to aretina, comprising: (a) delivering to a retinal neuron a nucleic acidexpression vector that encodes a type 1 rhodopsin domain of ared-shifted channelrhodopsin derived from algae, expressible in theretinal neuron; said vector comprising an open reading frame encodingthe type 1 rhodopsin domain of a red-shifted channelrhodopsinoperatively linked to a promoter sequence, and optionally, atranscriptional regulatory sequence; and (b) expressing said vector insaid retinal neuron, wherein the expressed rhodopsin renders saidretinal neuron photosensitive, thereby restoring light sensitivity tosaid retina or a portion thereof. In some embodiments, the type 1rhodopsin domain is encoded by SEQ ID NOS: 1-3.

In some embodiments, are methods of restoring light sensitivity to aretina, comprising: (a) delivering to a retinal neuron a nucleic acidexpression vector that encodes a rhodopsin domain of a red-shiftedchannelrhodopsin derived from algae, expressible in the retinal neuron;said vector comprising an open reading frame encoding the rhodopsindomain of a red-shifted channelrhodopsin operatively linked to apromoter sequence, and optionally, a transcriptional regulatorysequence; and (b) expressing said vector in said retinal neuron, whereinthe expressed rhodopsin renders said retinal neuron photosensitive,thereby restoring light sensitivity to said retina or a portion thereof.In some embodiments, the type 1 rhodopsin domain is encoded by SEQ IDNO: 4.

In some embodiments, are methods of restoring photosensitivity to aretina of a subject suffering from vision loss or blindness in whomretinal photoreceptor cells are degenerating or have degenerated anddied, said method comprising: (a) delivering to the retina of saidsubject a nucleic acid vector that encodes a type 1 rhodopsin domain ofa red-shifted channelrhodopsin derived from algae expressible in aretinal neuron; said vector comprising an open reading frame encodingthe type 1 rhodopsin domain of a red-shifted channelrhodopsinoperatively linked to a promoter sequence, and optionally, atranscriptional regulatory sequence; and (b) expressing said vector insaid retinal neuron, wherein the expressed rhodopsin renders saidretinal neuron photosensitive, thereby restoring photosensitivity tosaid retina or a portion thereof. In some embodiments, the type 1rhodopsin domain is encoded by SEQ ID NOS: 1-3.

In some embodiments, are methods of restoring photosensitivity to aretina of a subject suffering from vision loss or blindness in whomretinal photoreceptor cells are degenerating or have degenerated anddied, said method comprising: (a) delivering to the retina of saidsubject a nucleic acid vector that encodes a rhodopsin domain of ared-shifted channelrhodopsin derived from algae expressible in a retinalneuron; said vector comprising an open reading frame encoding therhodopsin domain of a red-shifted channelrhodopsin operatively linked toa promoter sequence, and optionally, a transcriptional regulatorysequence; and (b) expressing said vector in said retinal neuron, whereinthe expressed rhodopsin renders said retinal neuron photosensitive,thereby restoring photosensitivity to said retina or a portion thereof.In some embodiments, the type 1 rhodopsin domain is encoded by SEQ IDNO: 4.

In some embodiments, are any of the disclosed methods, wherein the type1 rhodopsin domain of a red-shifted channelrhodopsin having the aminoacid sequence of all or part of SEQ ID NOS: 1, 2 or 3, or a biologicallyactive fragment thereof that retains the biological activity of theencoded type 1 rhodopsin domain of a red-shifted channelrhodopsin or abiologically active conservative amino acid substitution variant of SEQID NOS: 1, 2 or 3 or of said fragment.

In some embodiments, are any of the disclosed methods, wherein therhodopsin domain of a red-shifted channelrhodopsin having the amino acidsequence of all or part of SEQ ID NO: 4, or a biologically activefragment thereof that retains the biological activity of the encodedrhodopsin domain of a red-shifted channelrhodopsin or a biologicallyactive conservative amino acid substitution variant of SEQ ID NO: 4 orof said fragment.

In some embodiments, are any of the disclosed methods wherein theexpression vectors include, but are not limited to, AAV viral vector. Insome embodiments, are any of the disclosed methods wherein the promoteris a constitutive promoter. In some embodiments, are any of thedisclosed methods wherein the constitutive promoter includes, but is notlimited to, a CMV promoter or a hybrid CMV enhancer/chicken β-actin(CAG) promoter. In some embodiments, are any of the disclosed methodswherein the promoter includes, but is not limited to, an inducibleand/or a cell type-specific promoter.

In some embodiments, a method of treating a neuronal disorder comprises:(a) delivering to a target neuron a nucleic acid expression vector thatencodes a type 1 rhodopsin domain of a red-shifted channelrhodopsinderived from algae, expressible in said target neuron; said vectorcomprising an open reading frame encoding the type 1 rhodopsin domain ofa red-shifted channelrhodopsin operatively linked to a promotersequence, and optionally, transcriptional regulatory sequences; and (b)expressing the expression vector in the target neuron, wherein theexpressed channelrhodopsin activates the target neuron upon exposure tolight. In some embodiments an above-described expression vector alsocomprises one or more transcriptional regulatory sequences operablylinked to the promoter and type 1 rhodopsin domain sequences. In someembodiments, the type 1 rhodopsin domain of a red-shiftedchannelrhodopsin has the amino acid sequence of all or part of SEQ IDNOS: 1, 2 or 3 and the rhodopsin domain sequences of SEQ ID NO:4, or abiologically active fragment thereof that retains the biologicalactivity of the encoded rhodopsin domain of a channelrhodopsin or is abiologically active conservative amino acid substitution variant of SEQID NOS: 1, 2, 3 or 4 or of said fragment. In some embodiments, theexpression vector comprises an AAV viral vector. In some embodiments,the promoter is a constitutive promoter. In some embodiments, theconstitutive promoter is a CMV promoter or a hybrid CMV enhancer/chickenβ-actin (CAG) promoter. In some embodiments, the promoter is aninducible and/or a cell type-specific promoter.

In some embodiments, a method of restoring light sensitivity to a retinacomprises: (a) delivering to a retinal neuron in a subject a nucleicacid expression vector that encodes a type 1 rhodopsin domain of ared-shifted channelrhodopsin derived from algae, expressible in theretinal neuron; said expression vector comprising an open reading frameencoding the type 1 rhodopsin domain of a red-shifted channelrhodopsinoperatively linked to a promoter sequence, and optionally, one or moretranscriptional regulatory sequences; and (b) expressing the expressionvector in the retinal neuron, wherein the expressed rhodopsin rendersthe retinal neuron photosensitive, thereby restoring light sensitivityto the retina or a portion thereof. In some embodiments, the type 1rhodopsin domain of a red-shifted channelrhodopsin has the amino acidsequence of all or part of SEQ ID NOS: 1-3 and therhodopsin domainsequences of SEQ ID NO: 4, or a biologically active fragment thereofthat retains the biological activity of the encoded type 1 rhodopsindomain of a red-shifted channelrhodopsin or is a biologically activeconservative amino acid substitution variant of SEQ ID NOS: 1-3 and therhodopsin domain sequences of SEQ ID NO: 4, or of said fragment. In someembodiments, the expression vector comprises an AAV viral vector. Insome embodiments, the promoter is a constitutive promoter. In someembodiments, the constitutive promoter is a CMV promoter or a hybrid CMVenhancer/chicken β-actin (CAG) promoter. In some embodiments, thepromoter is an inducible and/or a cell type-specific promoter.

In some embodiments, a method of restoring photosensitivity to a retinaof a subject suffering from vision loss or blindness in whom retinalphotoreceptor cells are degenerating or have degenerated and diedcomprises: (a) delivering to the retina of the subject a nucleic acidexpression vector that encodes a type 1 rhodopsin domain of ared-shifted channelrhodopsin derived from algae expressible in retinalneurons; said expression vector comprising an open reading frameencoding the type 1 rhodopsin domain of a red-shifted channelrhodopsinoperatively linked to a promoter sequence, and optionally,transcriptional regulatory sequences; and (b) expressing the expressionvector in the retinal neuron, wherein the expression of the rhodopsinrenders the retinal neuron photosensitive, thereby restoringphotosensitivity to said retina or a portion thereof. In someembodiments, the type 1 rhodopsin domain of a red-shiftedchannelrhodopsin has the amino acid sequence of all or part of SEQ IDNOS: 1-3 and the rhodopsin domain sequences of SEQ ID NO: 4, or abiologically active fragment thereof that retains the biologicalactivity of the encoded type 1 rhodopsin domain of a red-shiftedchannelrhodopsin or is a biologically active conservative amino acidsubstitution variant of SEQ ID NOS: 1-3 and the rhodopsin domainsequences of SEQ ID NO: 4, or of said fragment. In some embodiments, theexpression vector comprises an AAV viral vector. In some embodiments,the promoter is a constitutive promoter. In some embodiments, theconstitutive promoter is a CMV promoter or a hybrid CMV enhancer/chickenβ-actin (CAG) promoter. In other embodiments, the promoter is aninducible and/or a cell type-specific promoter.

To identify algal species were screened for candidates of newchannelrhodopsins with desirable characteristics, using thephotoelectrophysiological population assay for recordingrhodopsin-mediated photocurrents. EST and homology cloning was also usedto identify new channelopsin sequences in several algal species.

Exemplified below are the specifics of the process. MChR1 (SEQ ID NO: 1)which was derived from channelrhodopsin 1 of Mesostigma viride (SEQ IDNOS: 5 and 6, EMBL entry JF922293.1); CaChR1 (SEQ ID NO: 2) which wasderived from channelrhodopsin 1 of Chlamydomonas augustae (SEQ ID NO: 7and 8, EMBL entry JN596951.1; and CyChR1 (SEQ ID NO: 3) which wasderived from channelrhodopsin 1 Chlamydomonas yellowstonensis (SEQ IDNOS: 9 and 10, see EMBL entry JN596948.1) were cloned and expressed andare established as proteins having the most red-shifted spectralsensitivities so far reported, matches or surpasses knownchannelrhodopsins' channel kinetics, undergoes minimal inactivation uponsustained illumination, and exhibits pH-independent spectral sensitivityof membrane depolarization. This combination of properties makes MChR1(SEQ ID NO: 1), CaChR1 (SEQ ID NO: 2) and CyChR1 (SEQ ID NO: 3)particularly useful as a more precise and versatile agent for control ofneuronal activity as well as for optogenetic uses.

In some embodiments, the cloning and analysis of new channelopsins froma phylogenetically different alga expands the set of the currentlyavailable optogenetic techniques by introducing a fast, red-shifted andlittle-inactivated channelrhodopsin species, and also contributes to ourunderstanding of the sequence determinants of channelrhodopsin function.Furthermore, retinal neurons not normally sensitive to direct lightlocated in the retinas of blind mice, such as retinal ganglion cells(RGCs) and bipolar cells, can respond to light when a green algaeprotein called channelrhodopsin-2 (ChR2), or a biologically activefragment or a conservative amino acid substitution variant thereof, isinserted into the neuronal cell membranes. In some embodiments thedescribed channelrhodopsins may be used to transform retinal neurons notnormally sensitive to direct light located in the retinas. In someembodiments, are methods and compositions of a novel channelrhodopsin 2domain, identified as CraChr (CrChR) (SEQ ID NO: 4) which was derivedfrom channelopsin 2 of Chlamydomonas raudensis (SEQ ID NOS: 11 and 12,EMBL entry JN596949.1).

In some embodiments, molecular engineered variants (some with improvedactivity) of the described red-shifted channelrhodopsins bysite-specific mutagenesis and chimera construction. In some embodiments,the channelrhodopsins serve as receptors for phototaxis and thephotophobic response. Their photoexcitation initiates depolarization ofthe cell membrane.

In some embodiments, the type 1 rhodopsin domains of severalchannelrhodopsins were cloned and determined to have channel activitywhen they were expressed in mammalian HEK293 cells. Using these methodsnew channelrhodopsin variants, were determined to have improvedproperties with regards to, among other things, optogenetics. It has amore red-shifted spectral sensitivity at neutral pH than the previouslyavailable most red-shifted VChR1, and surpasses VChR1 by faster currentkinetics and smaller inactivation, all of which makes these type 1rhodopsin domains better suited for, among other things, rapid controlof neuronal activity.

The presently disclosed compositions and methods exemplified herein,demonstrate that that screening flagellate species by measuringrhodopsin-mediated photoelectric currents in vivo is an efficient methodfor identifying source organisms from which new channelrhodopsins withcharacteristics desirable for optogenetic applications can be isolated,cloned and expressed. Such screening cannot be performed by measuringphototaxis itself, i.e., the behavioral response, because even if analga demonstrates phototaxis, it is not necessarily mediated by achannelrhodopsin. As evidenced by a well-known example is Euglena, whichuses a flavin-binding adenylyl cyclase as a receptor for light controlof motility. The properties of properties of the cell'schannelrhodopsins can only be probed in algal cells by measuringphotoelectric responses.

Several channelrhodopsins were identified and characterized. MChR1 (SEQID NO: 1) which was derived from Mesostigma viride; CaChR1 (SEQ ID NO:2) which was derived from Chlamydomonas augustae; and CyChR1 (SEQ ID NO:3) which was derived from Chlamydomonas yellowstonensis.

MChR1 is a channelrhodopsin that was cloned from the flagellate alga M.viride identified as a candidate species by the above approach. WhenMChR1 was expressed in HEK cells it was determined that: (a) it exhibitsthe most red-shifted absorption among all studied channelrhodopsins; (b)its current kinetics is significantly faster than that of the otherknown red-shifted channelrhodopsin VChR1; (c) it showed lowerinactivation than VChR1 when stimulated with light of the samewavelength; and (d) the spectral sensitivity of channel activity waspH-independent. These four qualities are highly desirable for anoptogenetic tool intended for high-frequency control of rapid neuronalspiking.

MChR1 is expected to retain its excellent properties when expressed inneurons, because it has been shown earlier that photocurrents generatedby channelrhodopsins in HEK cells were quantitatively indistinguishablefrom those generated in neurons using channelrhodopsin-2 (30).

The utility of MChR1 and VChR1 for neurologic applications wasestablished by measuring their responses to high-frequency stimulationin mammalian HEK cells. At 25 Hz stimulus frequency the photocurrentgenerated by MChR1 was modulated to at least 50% amplitude, whereas thatgenerated by VChR1 showed practically no modulation (as demonstrated inFIG. 15). The much higher degree of amplitude modulation in MChR1, ascompared to VChR1, derives not only from the faster current decay, butalso from the significantly less inactivation of the photocurrent by aseries of light pulses (as demonstrated in FIG. 15). Consequently, atthe level of 50% amplitude modulation MChR1 demonstrated ˜15-fold betterfrequency response than VChR1 (as demonstrated in FIG. 16). In nativecells, the faster photocycle of MChR1, as compared to VChR1, is betteradapted for the higher rotation frequency of the unicellular M. viridethan that of colonial V. carteri.

The MChR1 sequence significantly expands channelrhodopsin diversity, asit deviates from other known channelrhodopsins in several importantaspects, which refines the sequence criteria for light-gated channelactivity.

Sequence analysis of the previously known four channelrhodopsin homologsidentified an array of five Glu residues in the predicted secondtransmembrane helix as a conserved structural feature suggested tocontribute to forming a water-containing channel. This hypothesis wastested by analysis of ChR2 mutants in which each individual Glu residuewas replaced by Ala.

Photocurrents generated by all these mutants were affected in eitheramplitude, kinetics, or inactivation, confirming their involvement inchannel function in ChR2. However, while the use of the current methodsand compositions should not be limited by any particular theory, resultsindicate that two of these Glu residues (corresponding to E83 and E97 inthe ChR2 sequence) are not required for channel activity at least insome channelrhodopsins, because (a) in MChR1, which behaves as a typicalchannelrhodopsin in HEK cells, they are replaced by non-carboxylatedresidues; and (b) their introduction in MChR1 actually inhibitedphotocurrents. Similarly, it has been suggested from analysis of thefour previously known channelrhodopsins that the presence of a Hisresidue at the position of the proton donor (D95 in BR) may befunctionally important. MChR1 lacks this feature since it contains Alaat this position, but substitution by His or Arg at this site each ledto suppression of channel activity. This amino acid position may besignificant but the presence of His at this site is not required forchannelrhodopsin functionality. On the other hand, residues that formthe predicted hydrogen-bond between putative helices C and D (C128 andD156 according to ChR2 numbering) are conserved in MChR1. MChR1 lacks aGlu residue at the position of E87 in ChR1, and its spectral sensitivityis independent of external pH.

Photocurrents generated by CaChR1 and CyChR1 differ from that of ChR1 intheir kinetics, inactivation and light dependence. The dependence of thespectral sensitivity of CaChR1- and CyChR1-generated currents on theexternal pH are consistent with the role of the residue in the positionof Glu87 (ChR1 numbering) in color tuning, as both these proteins alsocontain a Glu residue in this position (Glu94 and Glu95, respectively).In contrast, MChR1, which contains a non-carboxylated residue in thisposition, does not show the spectral shift over the entire tested pHrange from 5.3 to 9. This residue was suggested to contribute to theformation of a trimodal counterion of the protonated Schiff basecharacteristic of ChR1-like channelrhodopsins in contrast to ChR2-likeones. Therefore, protonation/deprotonation of this residue would beanticipated to alter the chromophore polarity and change the absorptionspectrum. Interestingly, in both CaChR1 and CyChR1 the spectraltransition occurs at a pH several units higher than that in ChR1.Moreover, CaChR1 and CyChR1 differ from the earlier known ChR1 in thatthe spectral maxima of their protonated forms is at 520 nm, compared to497 nm of ChR1. The red-shifted spectrum of VChR1, peaking at 530 nm,has been attributed to the residues in the positions of Ser141 andAla215 (BR numbering), which differentiates this protein from all otherknown channelrhodopsins. However, both CaChR1 and CyChR1 contain Gly andSer, respectively, in these positions, as do all other knownchannelopsins except VChR1, i.e., their red-shifted spectral sensitivitymust be due to other structural reasons. This result suggests theinvolvement of additional structural factors defining the pH dependenceof the spectral sensitivity.

Comparative sequence analysis reveals that both CaChR1 and CyChR1 differfrom ChR1 and, in fact, from all other reported channelrhodopsins, inthe residues found in the positions of Glu194 and Glu204 (BR numbering).Glu194 is conserved in all prior reported channelopsins, except CaChR1and CyChR1, in which it is replaced by Ser. Conversely, the position ofGlu204 is occupied by Ser in all previously described channelopsins, butit is Asp in CaChR1 and CyChR1. In BR, Glu194 and Glu204 are part of theextracellular hydrogen-bonded network that forms the proton releasepathway and are known to contribute to the pH dependence of spectraltransitions. These residues may also play a role in regulation of the pHdependence and/or color tuning in channelrhodopsins. Replacement of Serwith Glu at the site corresponding to Glu204 in BR led to the totalabolishment of channel activity in MChR1, suggesting the importance ofthis site for channelrhodopsin function.

One of the major challenges for optogenetic applications, especially inliving animals, are scattering of the stimulating light by biologicaltissues and its absorption by hemoglobin. Optogenetic tools withlong-wavelength absorption would exhibit minimal light attenuation fromthese effects, but most microbial rhodopsins do not fall into thiscategory. For instance, the absorption maximum of ChR2, which possessesseveral other useful properties and is thereby most frequently used as adepolarizing tool in optogenetics, is 470 nm. Several approaches havebeen taken to attempt to acquire red-shifted variants to reduce thelight-attenuation by scatter and absorption in tissue: (i) searching fornatural red-shifted channelrhodopsin variants in different algae (suchas those described herein); (ii) chimera construction; and (iii)site-directed mutagenesis. All of these approaches have in commonmodification of the apoprotein, and all have proved somewhat successful,although in some cases a desired spectral shift was accompanied bynegative effects such as slowing down of the current kinetics, or adecrease in the current amplitude.

Long-wavelength absorption by optogenetic tools is desirable to increasethe penetration depth of the stimulus light by minimizing tissuescattering and absorption by hemoglobin. In some embodiments, thelong-wavelength sensitivity of optogenetic microbial rhodopsins isenhanced using 3,4-Dehydroretinal (A2 retinal). A2 retinal(3,4-dehydroretinal) is a natural retinoid, its 11-cis form being foundin photoreceptor cells of certain invertebrates, fish and amphibians,where it may constitute the only retinal, or an additional chromophoreto A1 retinal. The presence of an additional double bond in the β-iononering of the chromophore results in pigments that absorb light at longerwavelengths, as compared to those formed with A1 (regular) retinal.Variations in A1/A2 ratio cause natural adaptive tuning of spectralsensitivity of vision in the organisms during adaptation to externalconditions. Reconstitution of bleached microbial rhodopsins(bacteriorhodopsin, halorhodopsin, sensory rhodopsins I and II) in vitrowith all-trans A2 retinal also shifts their absorption spectra to longerwavelengths. In some embodiments, spectral properties of optogenetictools were modified by incorporation of all-trans A2 retinal. Theaddition of A2 retinal, both ion pumps and channelrhodopsins formfunctional pigments with significantly red-shifted absorption. Despitethe presence of residual endogenous A1 retinal in HEK293 cells, largeextension of spectral sensitivity to longer wavelengths was observed.

In the examples below it is shown that A2 retinal reconstitutes a fullyfunctional archaerhodopsin-3 (AR-3) proton pump and fourchannelrhodopsin variants (CrChR1, CrChR2, CaChR1 and MvChR1).Substitution of A1 by A2 retinal significantly shifted the spectralsensitivity of all tested rhodopsins to longer wavelengths withoutaltering other aspects of their function. The spectral shift uponsubstitution of A1 by A2 in AR-3 was ˜1,000 cm⁻¹, close to that of otherlong-wavelength microbial rhodopsins. Notably, in all testedchannelrhodopsins the shifts were 1.4-1.8-fold larger than in othermicrobial or visual rhodopsins. In the case of CrChR1, binding of A2retinal raised the pKa of a spectral-tuning carboxylate residue, whichcontributed to the overall spectral shift.

In some embodiments, the long-wavelength sensitivity of optogeneticmicrobial rhodopsins is enhanced using 3,4-Dehydroretinal (A2 retinal).

In some embodiments, chromophore substitution provides a complementarystrategy to improve the efficiency of optogenetic tools. A2 retinalreconstitutes a fully functional archaerhodopsin-3 (AR-3) proton pumpand four channelrhodopsin variants (CrChR1, CrChR2, CaChR1 and MvChR1).Substitution of A1 by A2 retinal significantly shifted the spectralsensitivity of all tested rhodopsins to longer wavelengths withoutaltering other aspects of their function. The spectral shift uponsubstitution of A1 by A2 in AR-3 was ˜1,000 cm⁻¹, close to that of otherlong-wavelength microbial rhodopsins. Notably, in all testedchannelrhodopsins the shifts were 1.4-1.8-fold larger than in othermicrobial or visual rhodopsins. In the case of CrChR1, binding of A2retinal raised the pKa of a spectral-tuning carboxylate residue, whichcontributed to the overall spectral shift.

Channelrhodopsin Amino Acid Sequences:

The peptide amino acid sequences that can be used in various embodimentsincluding the channelrhodopsin amino acid sequences described herein(SEQ ID NOS.: 1-4,6,8 and 10), as well as analogues and derivativesthereof and functional fragments such as but not limited to the type 1rhodopsin domain. In fact, in some embodiments the any desired peptideamino acid sequences encoded by particular nucleotide sequences can beused, as is the use of any polynucleotide sequences encoding all, or anyportion, of desired peptide amino acid sequences. The degenerate natureof the genetic code is well-known, and, accordingly, eachchannelrhodopsin peptide amino acid-encoding nucleotide sequence isgenerically representative of the well-known nucleic acid “triplet”codon, or in many cases codons, that can encode the amino acid. As such,as contemplated herein, the channelrhodopsin peptide amino acidsequences described herein, when taken together with the genetic code(see, e.g., “Molecular Cell Biology,” Table 4-1 at page 109 (Darnell etal., eds., W. H. Freeman & Company, New York, N.Y., 1986)), aregenerically representative of all the various permutations andcombinations of nucleic acid sequences that can encode such amino acidsequences.

Such functionally equivalent peptide amino acid sequences (conservativesubstitutions) include, but are not limited to, additions orsubstitutions of amino acid residues within the amino acid sequencesencoded by a nucleotide sequence, but that result in a silent change,thus producing a functionally equivalent gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example: nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

Conservative amino acid substitutions may alternatively be made on thebasis of the hydropathic index of amino acids. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5). The use of thehydropathic amino acid index in conferring interactive biologicalfunction on a protein is understood in the art (Kyte and Doolittle, J.Mol. Biol. 157:105-132, 1982). It is known that in certain instances,certain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments the substitution of amino acids whose hydropathic indicesare within ±2 is included, while in other embodiments amino acidsubstitutions that are within ±1 are included, and in yet otherembodiments amino acid substitutions within ±0.5 are included.

Conservative amino acid substitutions may alternatively be made on thebasis of hydrophilicity, particularly where the biologically functionalprotein or peptide thereby created is intended for use in immunologicalembodiments. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein. Thefollowing hydrophilicity values have been assigned to these amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine(−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) andtryptophan (−3.4). In making changes based upon similar hydrophilicityvalues, in certain embodiments the substitution of amino acids whosehydrophilicity values are within ±2 is included, in certain embodimentsthose that are within ±1 are included, and in certain embodiments thosewithin ±0.5 are included.

Fusion Proteins:

The use of fusion proteins in which a polypeptide or peptide, or atruncated or mutant version of peptide is fused to an unrelated protein,polypeptide, or peptide, and can be designed on the basis of the desiredpeptide encoding nucleic acid and/or amino acid sequences describedherein. Such fusion proteins include, but are not limited to: IgFcfusions, which stabilize proteins or peptides and prolong half-life invivo; fusions to any amino acid sequence that allows the fusion proteinto be anchored to the cell membrane; or fusions to an enzyme,fluorescent protein, or luminescent protein that provides a markerfunction.

In certain embodiments, a fusion protein may be readily purified byutilizing an antibody that selectively binds to the fusion protein beingexpressed. In alternate embodiments, a fusion protein may be purified bysubcloning peptide encoding nucleic acid sequence into a recombinationplasmid, or a portion thereof, is translationally fused to anamino-terminal (N-terminal) or carboxy-terminal (C-terminal) tagconsisting of six histidine residues (a “His-tag”; see, e.g., Janknechtet al., Proc. Natl. Acad. Sci. USA 88:8972-8976, 1991). Extracts fromcells expressing such a construct are loaded onto Ni²⁺ nitriloaceticacid-agarose columns, and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

Recombinant Expression:

While the desired peptide amino acid sequences described can bechemically synthesized (see, e.g., “Proteins: Structures and MolecularPrinciples” (Creighton, ed., W. H. Freeman & Company, New York, N.Y.,1984)), large polypeptides sequences may advantageously be produced byrecombinant DNA technology using techniques well-known in the art forexpressing nucleic acids containing a nucleic acid sequence that encodesthe desired peptide. Such methods can be used to construct expressionvectors containing peptide encoding nucleotide sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination (see, e.g., “MolecularCloning, A Laboratory Manual,” supra, and “Current Protocols inMolecular Biology,” supra). Alternatively, RNA and/or DNA encodingdesired peptide encoding nucleotide sequences may be chemicallysynthesized using, for example, synthesizers (see, e.g.,“Oligonucleotide Synthesis: A Practical Approach” (Gait, ed., IRL Press,Oxford, United Kingdom, 1984)).

A variety of host-expression vector systems may be utilized to expresspeptide encoding nucleotide sequences. When the desired peptide orpolypeptide is soluble or a soluble derivative, the peptide orpolypeptide can be recovered from the host cell culture, i.e., from thehost cell in cases where the peptide or polypeptide is not secreted, andfrom the culture media in cases where the peptide or polypeptide issecreted by the host cell. However, suitable expression systems alsoencompass engineered host cells that express the desired polypeptide orfunctional equivalents anchored in the cell membrane. Purification orenrichment of the desired peptide from such expression systems can beaccomplished using appropriate detergents and lipid micelles, andmethods well-known to those skilled in the art. Furthermore, suchengineered host cells themselves may be used in situations where it isdesired not only to retain the structural and functional characteristicsof the peptide, but to assess biological activity, e.g., in certain drugscreening assays.

In certain applications, transient expression systems are desired.However, for long-term, high-yield production of recombinant proteins orpeptides, stable expression is generally preferred. For example, celllines that stably express the desired protein, polypeptide, peptide, orfusion protein may be engineered. Rather than using expression vectorsthat contain viral origins of replication, host cells can be transformedwith DNA controlled by appropriate expression control elements (e.g.,promoter, enhancer sequences, transcription terminators, polyadenylationsites, etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells are allowed to grow for about 1-2 days inan enriched media, and then switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection, and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci, which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines that express the desired gene products or portionsthereof. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of a desired protein, polypeptide or peptide.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223-232, 1977), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:2026-2034,1962), and adenine phosphoribosyltransferase (Lowy et al., Cell22:817-823, 1980) genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻cells, respectively. Anti-metabolite resistance can also be used as thebasis of selection for the following genes: dihydrofolate reductase(dhfr), which confers resistance to methotrexate (Wigler et al., Proc.Natl. Acad. Sci. USA 77:3567-3570, 1980, and O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527-1531, 1981); guanine phosphoribosyl transferase(gpt), which confers resistance to mycophenolic acid (Mulligan and Berg,Proc. Natl. Acad. Sci. USA 78:2072-2076, 1981); neomycinphosphotransferase (neo), which confers resistance to the aminoglycosideG-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14, 1981); andhygromycin B phosphotransferase (hpt), which confers resistance tohygromycin (Santerre et al., Gene 30:147-156, 1984).

Host cells/expression systems that may be used for purpose of providingcompositions to be used in the disclosed methods include, but are notlimited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis)transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmidDNA expression vector containing a desired peptide encoding nucleotidesequence; yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris)transformed with a recombinant yeast expression vector containing adesired peptide encoding nucleotide sequence; insect cell systemsinfected with a recombinant virus expression vector (e.g., baculovirus)containing a desired peptide encoding nucleotide sequence; plant cellsystems infected with a recombinant virus expression vector (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV), ortransformed with a recombinant plasmid expression vector (e.g., Tiplasmid), containing a desired peptide encoding nucleotide sequence; ormammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring arecombinant expression construct containing a desired peptide encodingnucleotide sequence and a promoter derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter, the vaccinia virus 7.5K promoter).

In bacterial systems, a number of different expression vectors may beadvantageously selected depending upon the use intended for the desiredgene product being expressed. For example, when a large quantity of sucha protein is to be produced, such as for the generation ofpharmaceutical compositions comprising a desired peptide, or for raisingantibodies to the protein, vectors that direct the expression of highlevels of fusion protein products that are readily purified may bedesirable. Such vectors include, but are not limited to: the E. coliexpression vector pUR278 (Ruther and Müller-Hill, EMBO J. 2:1791-1794,1983), in which a desired peptide encoding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye and Inouye,Nucleic Acids Res. 13:3101-3110, 1985, and Van Heeke and Schuster, J.Biol. Chem. 264:5503-5509, 1989); and the like. pGEX vectors (GEHealthcare, Piscataway, N.J.) may also be used to express a desiredpeptide moiety as a fusion protein with glutathione S-transferase (GST).In general, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads, followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned desired peptide encoding gene product can be releasedfrom the GST moiety.

In an exemplary insect system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express a desiredpeptide encoding sequence. The virus grows in Spodoptera frugiperdacells. A desired peptide encoding sequence may be cloned individuallyinto a non-essential region (for example the polyhedrin gene) of thevirus, and placed under control of an AcNPV promoter (for example thepolyhedrin promoter). Successful insertion of a desired peptide encodingsequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). The recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted polynucleotide is expressed (see, e.g., Smith et al., J. Virol.46:584-593, 1983, and U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a desired peptide encoding nucleotide sequence may be ligated toan adenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric sequence may thenbe inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing desired peptide products in infectedhosts (see, e.g., Logan and Shenk, Proc. Natl. Acad. Sci. USA81:3655-3659, 1984). Specific initiation signals may also be requiredfor efficient translation of inserted desired peptide encodingnucleotide sequences. These signals include the ATG initiation codon andadjacent sequences. In some cases exogenous translational controlsignals, including, perhaps, the ATG initiation codon, may be provided.Furthermore, the initiation codon should be in phase with the readingframe of the desired peptide encoding coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see, e.g., Nevins, CRC Crit. Rev.Biochem. 19:307-322, 1986).

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review, see, e.g., “Current Protocols inMolecular Biology,” supra, Ch. 13, Bitter et al., Meth. Enzymol.153:516-544, 1987, “DNA Cloning,” Vol. II, Ch. 3 (Glover, ed., IRLPress, Washington, D.C., 1986); Bitter, Meth. Enzymol. 152:673-684,1987, “The Molecular Biology of the Yeast Saccharomyces: Life Cycle andInheritance” (Strathern et al., eds., Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1981), and “The Molecular Biology of the YeastSaccharomyces: Metabolism and Gene Expression” (Strathern et al., eds.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1982).

In plants, a variety of different plant expression vectors can be used,and expression of a desired peptide encoding sequence may be driven byany of a number of promoters. For example, viral promoters such as the35S RNA or 19S RNA promoters of CaMV (Brisson et al., Nature310:511-514, 1984), or the coat protein promoter of TMV (Takamatsu etal., EMBO J. 6:307-311, 1987) may be used. Alternatively, plantpromoters such as the promoter of the small subunit of RUBISCO (Coruzziet al., EMBO J. 3:1671-1679, 1984, and Broglie et al., Science224:838-843, 1984), or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B (Gurley et al., Mol. Cell. Biol. 6:559-565, 1986) may be used.These constructs can be introduced into plant cells using, for example,Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, or electroporation. For reviews of suchtechniques, see, e.g., Weissbach and Weissbach, in “Methods in PlantMolecular Biology,” Section VIII (Schuler and Zielinski, eds., AcademicPress, Inc., New York, N.Y., 1988), and “Plant Molecular Biology,”2^(nd) Ed., Ch. 7-9 (Grierson and Covey, eds., Blackie & Son, Ltd.,Glasgow, Scotland, United Kingdom, 1988).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted desired peptide encoding sequence, ormodifies and processes the desired peptide encoding nucleic acidsequence in a desired fashion. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products may affect certainfunctions of the protein. Different host cells have characteristic andspecific mechanisms for post-translational processing and modificationof proteins and peptides. Appropriate cell lines or host systems can bechosen to ensure the correct or desired modification and processing ofthe desired protein, polypeptide, or peptide expressed. To this end,eukaryotic host cells that possess the cellular machinery for desiredprocessing of the primary transcript, and glycosylation and/orphosphorylation of desired peptide encoding nucleic acid sequence beused. Such mammalian host cells include, but are not limited to, Chinesehamster ovary (CHO), VERO, baby hamster kidney (BHK), HeLa, monkeykidney (COS), MDCK, 293, 3T3, WI38, human hepatocellular carcinoma(e.g., Hep G2), and U937 cells.

Compositions as Therapeutics:

The use of channelrhodopsins, or active fragments thereof such as butnot limited to the type 1 rhodopsin domain as therapeutics. In certainembodiments the presently disclosed compositions and are used to improveoptogenetic techniques and applications as well as can be used to aid indiagnosis, prevention, and/or treatment of among other things neuronmediated disorders, neurologic disorders (such as Parkinson's disease)and as therapy for ocular disorders.

In certain embodiments the presently disclosed compositions can beadministered in combination with one or more additional compounds oragents (“additional active agents”) for the treatment, management,and/or prevention of among other things neuron mediated disorders,neurologic disorders (such as Parkinson's disease) and as therapy forocular disorders. Such therapies can be administered to a patient attherapeutically effective doses to treat or ameliorate, among otherthings, neuron mediated disorders, neurologic disorders (such asParkinson's disease) and as therapy for ocular disorders. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in any delay in onset, amelioration, or retardationof disease symptoms.

Toxicity and therapeutic efficacy of such compositions can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index, expressed as the ratio LD₅₀/ED₅₀. Compositionsthat exhibit large therapeutic indices are preferred. Compounds thatexhibit toxic side effects may be used in certain embodiments, however,care should usually be taken to design delivery systems that target suchcompositions preferentially to the site of affected tissue, in order tominimize potential damage to uninfected cells and, thereby, reduce sideeffects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosages for use in humans. The dosages of suchcompositions lie preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending on the dosage form employed and the route ofadministration utilized. For any composition, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test composition that achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Plasma levels may bemeasured, for example, by high performance liquid chromatography.

When the therapeutic treatment of among other things neurologicdisorders (such as Parkinson's disease) and as therapy for oculardisorders is contemplated, the appropriate dosage may also be determinedusing animal studies to determine the maximal tolerable dose, or MTD, ofa bioactive agent per kilogram weight of the test subject. In general,at least one animal species tested is mammalian. Those skilled in theart regularly extrapolate doses for efficacy and avoiding toxicity toother species, including human. Before human studies of efficacy areundertaken, Phase I clinical studies help establish safe doses.

Additionally, the bioactive agent may be coupled or complexed with avariety of well established compositions or structures that, forinstance, enhance the stability of the bioactive agent, or otherwiseenhance its pharmacological properties (e.g., increase in vivohalf-life, reduce toxicity, etc.).

Such therapeutic agents can be administered by any number of methodsknown to those of ordinary skill in the art including, but not limitedto, inhalation, subcutaneous (sub-q), intravenous (I.V.),intraperitoneal (I.P.), intramuscular (I.M.), or intrathecal injection,or topically applied (transderm, ointments, creams, salves, eye drops,and the like), as described in greater detail below.

Pharmaceutical Compositions:

Pharmaceutical compositions for use in accordance with the presentlydescribed compositions may be formulated in conventional manners usingone or more physiologically acceptable carriers or excipients.

The pharmaceutical compositions can comprise formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption or penetration of the composition.Suitable formulation materials include, but are not limited to: aminoacids (for example, glycine, glutamine, asparagine, arginine andlysine); antimicrobials; antioxidants (for example, ascorbic acid,sodium sulfite and sodium hydrogen-sulfite); buffers (for example,borate, bicarbonate, Tris-HCl, citrates, phosphates and other organicacids); bulking agents (for example, mannitol and glycine); chelatingagents (for example, ethylenediamine tetraacetic acid (EDTA));complexing agents (for example, caffeine, polyvinylpyrrolidone,beta-cyclodextrin, and hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides, disaccharides, and other carbohydrates (for example,glucose, mannose and dextrins); proteins (for example, serum albumin,gelatin and immunoglobulins); coloring, flavoring, and diluting agents;emulsifying agents; hydrophilic polymers (for example,polyvinylpyrrolidone); low molecular weight polypeptides; salt-formingcounterions (for example, sodium); preservatives (for example,benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbicacid and hydrogen peroxide); solvents (for example, glycerin, propyleneglycol and polyethylene glycol); sugar alcohols (for example, mannitoland sorbitol); suspending agents; surfactants or wetting agents (forexample, pluronics, PEG, sorbitan esters, polysorbates (for example,polysorbate 20 and polysorbate 80), triton, tromethamine, lecithin,cholesterol, and tyloxapal); stability enhancing agents (for example,sucrose and sorbitol); tonicity enhancing agents (for example, alkalimetal halides (for example, sodium or potassium chloride), mannitol, andsorbitol); delivery vehicles; diluents; excipients; and pharmaceuticaladjuvants (“Remington's Pharmaceutical Sciences,” 18^(th) Ed. (Gennaro,ed., Mack Publishing Company, Easton, Pa., 1990)).

Additionally, the described therapeutic peptides can be linked to ahalf-life extending vehicle. Certain exemplary half-life extendingvehicles are known in the art, and include, but are not limited to, theFc domain, polyethylene glycol, and dextran (see, e.g., PCT PatentApplication Publication No. WO 99/25044).

These agents may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The agents may also be formulated as compositions for rectaladministration such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

In addition to the formulations described previously, the agents mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. For example, agents maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil), ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.The compositions may, if desired, be presented in a pack or dispenserdevice, which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Active compositions can be administered by controlled release means orby delivery devices that are well-known to those of ordinary skill inthe art. Examples include, but are not limited to, those described inU.S. Pat. Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719,5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476,5,354,556, and 5,733,566. Such dosage forms can be used to provide slowor controlled-release of one or more active ingredients using, forexample, hydropropylmethyl cellulose, other polymer matrices, gels,permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof, toprovide the desired release profile in varying proportions. Exemplarysustained release matrices include, but are not limited to, polyesters,hydrogels, polylactides (see, e.g., U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058,481), copolymers of L-glutamicacid and gamma ethyl-L-glutamate (see, e.g., Sidman et al., Biopolymers22:547-556, 1983), poly (2-hydroxyethyl-methacrylate) (see, e.g., Langeret al., J. Biomed. Mater. Res. 15:167-277, 1981, and Langer, Chemtech12:98-105, 1982), ethylene vinyl acetate (Langer et al., supra), andpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may include liposomes,which can be prepared by any of several methods known in the art (see,e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692, 1985,and European Patent Application Publication Nos. EP 036,676, EP 088,046,and EP 143,949). Suitable controlled-release formulations known to thoseof ordinary skill in the art, including those described herein, can bereadily selected for use with the presently disclosed compositions.Certain embodiments encompass single unit dosage forms suitable for oraladministration such as, but not limited to, tablets, capsules, gelcaps,and caplets that are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving therapy over that achieved by their non-controlledcounterparts. Ideally, use of an optimally designed controlled-releasepreparation in medical treatment is characterized by a minimum of drugsubstance being employed to cure or control the condition in a minimumamount of time. Advantages of controlled-release formulations includeextended activity of the drug, reduced dosage frequency, and increasedpatient compliance. In addition, controlled-release formulations can beused to affect the time of onset of action or other characteristics,such as blood levels of the drug, and can thus affect the occurrence ofside (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of active ingredient that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof active ingredient to maintain this level of therapeutic orprophylactic effect over an extended period of time. In order tomaintain this relatively constant level of active ingredient in thebody, the drug must be released from the dosage form at a rate that willreplace the amount of active ingredient being metabolized and excretedfrom the body. Controlled-release of an active ingredient can bestimulated by various conditions including, but not limited to, pH,temperature, enzymes, water, or other physiological conditions orcompositions.

In some cases, active ingredients of the disclosed methods andcompositions are preferably not administered to a patient at the sametime or by the same route of administration. Therefore, in someembodiments are kits that, when used by the medical practitioner, cansimplify the administration of appropriate amounts of active ingredientsto a patient.

A typical kit comprises a single unit dosage form of one or more of thetherapeutic agents disclosed, alone or in combination with a single unitdosage form of another agent that may be used in combination with thedisclosed compositions. Disclosed kits can further comprise devices thatare used to administer the active ingredients. Examples of such devicesinclude, but are not limited to, syringes, drip bags, patches, andinhalers.

Disclosed kits can further comprise pharmaceutically acceptable vehiclesthat can be used to administer one or more active ingredients. Forexample, if an active ingredient is provided in a solid form that mustbe reconstituted for parenteral administration, the kit can comprise asealed container of a suitable vehicle in which the active ingredientcan be dissolved to form a particulate-free sterile solution that issuitable for parenteral administration. Examples of pharmaceuticallyacceptable vehicles include, but are not limited to: Water for InjectionUSP; aqueous vehicles such as, but not limited to, Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, and Lactated Ringer's Injection; water-misciblevehicles such as, but not limited to, ethyl alcohol, polyethyleneglycol, and polypropylene glycol; and non-aqueous vehicles such as, butnot limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyloleate, isopropyl myristate, and benzyl benzoate. However, in specificembodiments, the disclosed formulations do not contain any alcohols orother co-solvents, oils or proteins.

Channelrhodopsin Nucleic Acid Sequences:

Channelrhodopsin nucleic acid sequences for use in the disclosed methodsand compositions include, but are not limited to, the active portion ofthe presently disclosed algal derived red-shifted channelrhodopsins (SEQID NOS: 5, 7 and 9), including but not limited to those described, suchas but not limited to the nucleic acid sequences that encode the type 1rhodopsin domain, an active portion of the presently disclosed algalderived red-shifted channelrhodopsins, such as but not limited to thetype 1 or rhodopsin domains disclosed (SEQ ID NOS: 1, 2, 3 or 4)

In some embodiments, the use of an active portion of a presentlydisclosed algal derived red-shifted channelrhodopsin, such as but notlimited to the type 1 rhodopsin domain, includes all or portions of thesequences described herein (and expression vectors comprising the same),and additionally contemplates the use of any nucleotide sequenceencoding a contiguous an active portion of the presently disclosed algalderived red-shifted channelrhodopsins, such as but not limited to thetype 1 rhodopsin domain, open reading frame (ORF) that hybridizes to acomplement of a channelrhodopsin or channelopsin sequence describedherein under highly stringent conditions, e.g., hybridization tofilter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mMEDTA at 65EC, and washing in 0.1×SSC/0.1% SDS at 68EC (“CurrentProtocols in Molecular Biology,” Vol. 1 and 2 (Ausubel et al., eds.,Green Publishing Associates, Incorporated, and John Wiley & Sons,Incorporated, New York, N.Y., 1989)), and encodes a functionallyequivalent channelrhodopsin (or active portion thereof, such as but notlimited to the type 1 rhodopsin domain) gene product or the activeportion thereof. Additionally contemplated is the use of any nucleotidesequence that hybridizes to the complement of a DNA sequence thatencodes a channelrhodopsin amino acid sequence under moderatelystringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42EC(“Current Protocols in Molecular Biology,” supra), yet still encodes afunctionally equivalent channelrhodopsin product. Functional equivalentsof channelrhodopsin include, but are not limited to, naturally occurringversions of channelrhodopsin present in other species (orthologs andhomologs), and mutant versions of channelrhodopsin, whether naturallyoccurring or engineered (by site directed mutagenesis, gene shuffling,or directed evolution, as described in, for example, U.S. Pat. No.5,837,458) or active portion thereof, such as but not limited to thetype 1 rhodopsin domain. The disclosure also includes the use ofdegenerate nucleic acid variants (due to the redundancy of the geneticcode) of the identified channelrhodopsin polynucleotide sequences.

Additionally contemplated is the use of polynucleotides encodingchannelrhodopsin ORFs, or their functional equivalents, encoded bypolynucleotide sequences that are about 99, 95, 90, or about 85 percentsimilar to the corresponding regions of the an algae derivedchannelrhodopsin sequences described herein (as measured by BLASTsequence comparison analysis using, for example, the University ofWisconsin GCG sequence analysis package (SEQUENCHER 3.0, Gene CodesCorporation, Ann Arbor, Mich.) using default parameters).

Transgenic Animals:

The present disclosure provides methods and compositions for thecreation and use of both human and non-human transgenic animals thatcarry an algae derived channelrhodopsin transgene in all their cells, aswell as non-human transgenic animals that carry an algae derivedchannelrhodopsin transgene in some, but not all their cells, for examplein certain neuronal cells. Human and non-human mammals of any species,including, but not limited to, mice, rats, rabbits, guinea pigs, pigs,micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, andchimpanzees, can be used to generate transgenic animals carrying analgae derived channelrhodopsin polynucleotide (and/or expressing analgae derived polypeptide) may be integrated as a single transgene or inconcatamers, e.g., head-to-head or head-to-tail tandems. An algaederived channelrhodopsin transgene may also be selectively introducedinto and activated in a particular cell-type (see, e.g., Lakso et al.,Proc. Natl. Acad. Sci. USA 89:6232-6236, 1992). The regulatory sequencesrequired for such a cell-type specific activation will depend upon theparticular cell-type of interest, and will be apparent to those of skillin the art.

Should it be desired that an algae derived channelrhodopsin, or fragmentthereof, transgene be integrated into the chromosomal site of theendogenous copy of the mammalian channelrhodopsin gene, gene targetingis generally preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous channelrhodopsin gene are designed for the purpose ofintegrating, via homologous recombination with chromosomal sequences,into and disrupting the function of the endogenous channelrhodopsin gene(i.e., “knock-out” animals). In this way, the expression of theendogenous channelrhodopsin gene may also be eliminated by insertingnon-functional sequences into the endogenous channelrhodopsin gene. Thetransgene may also be selectively introduced into a particularcell-type, thus inactivating the endogenous channelrhodopsin gene inonly that cell-type (see, e.g., Gu et al., Science 265:103-106, 1994).The regulatory sequences required for such a cell-type specificinactivation will depend upon the particular cell-type of interest, andwill be apparent to those of skill in the art.

Any technique known in the art may be used to introduce achannelrhodopsin, or fragment thereof, transgene into animals to producethe founder lines of transgenic animals. Such techniques include, butare not limited to: pronuclear microinjection (U.S. Pat. No. 4,873,191);retrovirus-mediated gene transfer into germ lines (van der Putten etal., Proc. Natl. Acad. Sci. USA 82:6148-6152, 1985); gene targeting inembryonic stem cells (Thompson et al., Cell 56:313-321, 1989);electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803-1814, 1983);sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723, 1989);and positive-negative selection, as described in U.S. Pat. No.5,464,764. For a review of such techniques, see, e.g., Gordon, Int. Rev.Cytol. 115:171-229, 1989.

Once transgenic animals have been generated, the expression of therecombinant channelrhodopsin gene, or fragment thereof, may be assayedutilizing standard techniques. Initial screening may be accomplished bySouthern blot analysis or PCR techniques to analyze animal tissues toassay whether integration of the channelrhodopsin transgene has takenplace. The level of mRNA expression of the channelrhodopsin transgene inthe tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof cell-type samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of an algae derivedchannelrhodopsin-expressing tissue can also be evaluatedimmunocytochemically using antibodies selective for the channelrhodopsintransgene product.

In certain embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors which encode a protein or peptide thatincludes within its amino acid sequence an amino acid sequence of achannelrhodopsin or a functional portions or variant thereof, such asthose identified and cloned: MChR1 (SEQ ID NO: 1), CaChR1 (SEQ ID NO:2), CyChR1 (SEQ ID NO: 3) and CraChR2 (SEQ ID NO: 4). In someembodiments, a portion of a channelrhodopsin and has relatively fewamino acids which are not identical to, or a biologically functionalequivalent of, the amino acids of the full-length channelrhodopsin. Theterm “functional equivalent” is well understood in the art. Accordingly,sequences which have between about 70% and about 80%; or morepreferably, between about 85% and about 90%; or even more preferably,between about 90 and 95% and about 99%; of amino acids which areidentical or functionally equivalent to the amino acids of theidentified and cloned: MChR1 (SEQ ID NO: 1), CaChR1 (SEQ ID NO: 2),CyChR1 (SEQ ID NO: 3) or CraChR2 (SEQ ID NO:−4) will be sequences whichare essentially as set forth in SEQ ID NOS: 1-4.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with othersequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared whichinclude a short stretch complementary to nucleic acids that encode thepolypeptides of SEQ ID NOS: 1-4, such as about 10 to 15 or 20, 30, or 40or so nucleotides, and which are up to 2000 or so base pairs in length.DNA segments with total lengths of about 8000, 7000, 6000, 5000, 4000,3000, 2000, 1000, 500, 200, 100 and about 50 base pairs in length arealso contemplated to be useful.

In some embodiments, isolated nucleic acids that encode the amino acidsof a channelrhodopsin or fragment thereof and recombinant vectorsincorporating nucleic acid sequences which encode a channelrhodopsinprotein or peptide and that includes within its amino acid sequence anamino acid sequence in accordance with SEQ ID NOS: 1-4. In someembodiments, a purified nucleic acid segment that encodes a protein thatencodes a channelrhodopsin or fragment thereof, the recombinant vectormay be further defined as an expression vector comprising a promoteroperatively linked to said channelrhodopsin-encoding nucleic acidsegment.

In additional embodiments, is a host cell, made recombinant with arecombinant vector comprising channelrhodopsin-encoding nucleic acidsegments. The recombinant host cell may be a prokaryotic cell or aeukaryotic cell. As used herein, the term “engineered” or “recombinant”cell is intended to refer to a cell into which a recombinant gene, suchas a gene encoding a channelrhodopsin, has been introduced. Therefore,engineered cells are distinguishable from naturally occurring cellswhich do not contain a recombinantly introduced gene. Engineered cellsare thus cells having a gene or genes introduced through the hand ofman. Recombinantly introduced genes will either be in the form of a copyof a genomic gene or a cDNA gene, or will include genes positionedadjacent to a promoter not naturally associated with the particularintroduced gene. In some embodiments, nucleic acid molecules havingsequence regions consisting of contiguous nucleotide stretches of about14, 15-20, 30, 40, 50, or even of about 100 to about 200 nucleotides orso, identical or complementary to the channelrhodopsin-encoding nucleicacid sequences.

Transgene Based Therapies:

The nucleic acids sequences that encode an active portion of thepresently disclosed red-shifted channelrhodopsins, include but are notlimited to the type 1 rhodopsin domains identified in SEQ ID NOS: 1-3 orthe rhodopsin domain sequences of SEQ ID NO: 4.

In certain embodiments the presently disclosed compositions and are usedto improve optogenetic techniques and applications as well as can beused to aid in diagnosis, prevention, and/or treatment of neurologicdisorders, such as but not limited to Parkinson's disease, as well asfor ocular disorders.

In some embodiments, methods and compositions are used to identify andcharacterize multiple channelrhodopsins derived from algae. The cloningand expression of the type 1 rhodopsin domain of the channelrhodopsinsand expression in mammalian cells demonstrates that thesechannelrhodopsins have improved characteristics that can be used foroptogenetic applications as well as therapeutic agents.

For example, a disclosed method and composition may be used in, amongother things, retinal gene therapy for mammals (as described in, amongothers, U.S. Pat. Nos. 5,827,702, 7,824,869 and US Patent PublicationNumber 20100015095 as well as in WIPO publications WO 2000/15822 and WO1998/48097). A genetically engineered ocular cell is produced bycontacting the cell with an exogenous nucleic acid under conditions inwhich the exogenous nucleic acid is taken up by the cell for expression.The exogenous nucleic acid is described as a retrovirus, an adenovirus,an adeno-associated virus or a plasmid. Retinal gene transfer of areporter gene, green fluorescent protein (GFP), using a recombinantadeno-associated virus (rAAV) was demonstrated in normal primates(Bennett, J et al. 1999 Proc. Natl. Acad. Sci. USA 96, 9920-25). Therescue of photoreceptors using gene therapy in a model of rapiddegeneration of photoreceptors using mutations of the RP65 gene andreplacement therapy with the normal gene to replace or supplant themutant gene (See, for example, US Patent Publication No. 2004/0022766)has been used to treat a naturally-occurring dog model of severe diseaseof retinal degenerations—the RPE65 mutant dog, which is analogous tohuman LCA. By expressing photosensitive membrane-channels or moleculesin surviving retinal neurons of the diseased retina by viral based genetherapy method, the present invention may produce permanent treatment ofthe vision loss or blindness with high spatial and temporal resolutionfor the restored vision.

In some embodiments, introduction and expression of channelrhodopsins,such as those described herein, inocular neuronal cells, for example,impart light sensitivity to such retinas and restoring one or moreaspects of visual responses and functional vision to a subject sufferingfrom such degeneration. By restoring light sensitivity to a retinalacking this capacity, due to disease, a mechanism for the most basiclight-responses that are required for vision is provided. In someembodiments, the functional domains of channelrhodopsins, such as MChR1,CaChR1, CyChR1 and CrChR2 may be used to restore light sensitivity tothe retinas that have undergone rod and cone degeneration by expressingthe channelrhodopsin in inner retinal neurons in vivo. In someembodiments these channelrhodopsins may be introduced using techniquesthat include, but are not limited to, retinal implants, corticalimplants, lateral geniculate nucleus implants, or optic nerve implants

In some embodiments, the red-shifted channelrhodopsins are inserted intothe retinal neurons that survived after the rods and cones have died inan area or portion of the retina of a subject, using the transfer ofnucleic acids, alone or within an expression vector. Such expressionvectors may be constructed, for example, by introduction of the desirednucleic acid sequence into a virus system known to be of use for genetherapy applications, such as, but not limited to, AAV, retroviruses andalike.

In some embodiments the red-shifted channelrhodopsins may be insertedinto retinal interneurons. These cells then can become light sensitiveand send signals via the optic nerve and higher order visual pathways tothe visual cortex where visual perception occurs, as has beendemonstrated electrophysiologicly in mice. In some embodiments, amongother routes, intravitreal and/or subretinal injections may be used todeliver channelrhodopsin molecules or virus vectors expressing the same.

In some embodiments, the active portion of the presently disclosed algalderived red-shifted channelrhodopsins, such as but not limited to thetype 1 rhodopsin domain of these channelrhodopsins, can be used torestore light sensitivity to a retina, by delivering to retinal neuronsa nucleic acid expression vector that encodes algal derived red-shiftedchannelrhodopsins (such as but not limited to the type 1 rhodopsindomain of these channelrhodopsins) that is expressible in the neurons,which vector comprises an open reading frame encoding the rhodopsin, andoperatively linked thereto, a promoter sequence, and optionally,transcriptional regulatory sequences; and expressing the vector in theneurons, thereby restoring light sensitivity.

In certain embodiments the channel rhodopsin can be algal derivedred-shifted channelrhodopsins such as, but not limited to functionaldomains of channelrhodopsins, such as MChR1, CaChR1, CyChR1 and CrChR2,or a biologically active fragment or conservative amino acidsubstitution variant thereof, such as but not limited to the type 1rhodopsin domain. The vector system may be recombinant AAV, the promotermay be a constitutive promoter such as, but not limited to, a CMVpromoter or a hybrid CMV enhancer/chicken β-actin promoter (CAG).

The following Examples section provides further details regardingexamples of various embodiments. It should be appreciated by those ofskill in the art that the techniques disclosed in the examples thatfollow represent techniques and/or compositions discovered by theinventors to function well. However, those of skill in the art should,in light of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention. These examples are illustrations of the methods andsystems described herein and are not intended to limit the scope of theinvention. The type 1 rhodopsin domain of a channelrhodopsin derivedfrom Mesostigma viride, MChR1; CaChR1, the type 1 rhodopsin domain of achannelrhodopsin derived from Chlamydomonas augustae and CyChR1 the type1 rhodopsin domain of a channelrhodopsin derived from Chlamydomonasyellowstonensis as identified herein were chosen to exemplify how thesechannelrhodopsins can be identified, cloned, expressed, characterizedand used. Non-limiting examples of such include, but are not limited to

EXAMPLES Channelopsins Derived from Mesostigma viride Example 1 Sourceand Growth of Algae

Mesostigma viride strain CCMP2046 (aka NIES 296) was obtained from theProvasoli-Guillard National Center for the Culture of MarinePhytoplankton. Cells were grown at 25° C. in modified Jaworski medium(the concentration of Ca(NO₃)₂ was increased to 0.17 mM; 0.5 mM KN0₃,0.15 μM ZnS0₄ and 0.04 μM CoCl₂ were added) under a 12 h light (˜3,000lux): 12 h dark cycle.

Example 2 Rhodopsin-Mediated Photocurrents in Algae Cells

Currents were measured with the population assay described in(Sineshchekov, O. A., E. G. Govorunova, A. Der, L. Keszthelyi, and W.Nultsch. Photoelectric responses in phototactic flagellated algaemeasured in cell suspension. J. Photochem. Photobiol. B: Biol.13:119-134, 1992). Two platinum wires immersed in a cell suspension pickup an electrical current generated in response to a unilateralexcitation flash from a Vibrant HE 35511 Tunable Laser (OPOTEK Inc.,Carlsbad, Calif.) set at desired wavelengths. The signal was amplifiedby a low-noise current amplifier (Model 428, Keithley Instruments,Cleveland, Ohio) and digitized by a Digidata 1322A supported by pClamp10 software (both Molecular Devices, Union City, Calif.).

Example 3 Cloning and Expression of M. Viride Channelrhodopsin

Total RNA was extracted from 500 ml of 1 week-old culture of M. virideusing Trizol reagent (Invitrogen, Carlsbad, Calif.). Synthesis of 3′ and5′ RACE-ready first-strand cDNAs and 3′ and 5′ RACE PCR were carried outusing the SMARTer RACE cDNA amplification kit (Clontech Laboratories,Takara Bio Company, Mountain View, Calif.) using primers designedaccording to the opsin sequence fragment found in the TaxonomicallyBroad EST Database Taxonomically Broad EST Database (O'Brien et al.Nucleic Acids Res. January; 35 (Database issue): D445-D451, 2007). Theoverlapping RACE fragments were combined by fusion PCR, cloned intopCR2.1-TOPO vector (Invitrogen) and fully sequenced. The 7TM domain(encoding residues 1-331) was inserted between BamHI and NotI sites toreplace the VChR1 sequence in the pcDNA3.1/VChR1-EYFP mammalianexpression vector provided by K. Deisseroth (Stanford University). Thepresence of a fluorescent tag is not expected to affect channelrhodopsinproperties, as has been shown by quantitative comparison ofphotocurrents generated by YFP-, mCherry- and myc-tagged ChR2 (Nikolic,K., N. Grossman, M. S. Grubb, J. Burrone, C. Toumazou, and P. Degenaar.Photocycles of channelrhodopsin-2. Photochem. Photobiol. 85:400-411,2009). Point mutations were introduced using the QuikChange XLSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.). HEK293cells were transfected using the TransPass COS/293 transfection reagent(New England Biolabs, Ipswich, Mass.). All-trans-retinal (stock solutionin ethanol) was added to a final concentration 2.5 μM.

Example 4 Photoreceptor Activity

Whole-Cell Patch Clamp Recording:

Measurements were performed 24-48 h after transfection with an Axopatch200B amplifier (Molecular Devices, Union City, Calif.). The signals weredigitized with a Digidata 1440A using pClamp 10 software (both fromMolecular Devices). Patch pipettes with resistances 2-5 MΩ werefabricated from borosilicate glass and filled with the followingsolution (in mM): KCl 126, MgSO₄ 2, CaCl₂ 0.5, EGTA 5, glucose 20, HEPES25, pH 7.2. The bath solution contained (in mM): NaCl 150, CaCl₂ 1.8,KCl 4, MgCl₂ 1, glucose 5, HEPES 10, pH 7.4. Unless otherwise indicated,the holding potential was −60 mV. Light excitation was provided by aPolychrome IV light source (T.I.L.L. Photonics GMBH, Grafelfing,Germany) pulsed with a mechanical shutter (Uniblitz Model LS6, VincentAssociates, Rochester, N.Y.). The light intensity was attenuated withthe built-in Polychrome system or with neutral density filters.

Rhodopsin-Mediated Photoelectric Currents in M. viride Cells:

Photoreceptor currents in native M. viride cells were measured in cellsuspensions under unilateral laser flash excitation. The response wassimilar to those previously observed in other phototactic flagellateswith the intra-chloroplast eyespots. However, it was significantlyfaster, with a peak time of <0.5 ms and a half-rise time of 100 μs (asshown in FIG. 1). In addition, photocurrents in M. viride lacked akinetic contribution of the late (delayed) photoreceptor currentdiscovered earlier in green algae to the recorded signal (as shown inFIG. 1). The second component was also not found in the fluence-responsedependence (as shown in FIG. 2), which was well fit with a singleexponential function that saturated at about the same light intensitiesas previously studied early photoreceptor currents in other flagellatealgae. However, in contrast to other flagellates, the curve in M. viridewas well fit without introducing an additional low-saturating component,which means that the amplified current component did not contribute tothe signals.

The action spectrum for the photoreceptor current in suspensions of M.viride cells is shown in FIG. 3. The spectral sensitivity was estimatedby calculating the mean value of the current response between 50 μs and2 ms. The first 50 μs was omitted because during this time a fastphotosynthetic signal developed. To completely eliminate thecontribution of this signal, the traces were further corrected bysubtracting the responses evoked by a 680-nm laser flash. Thiswavelength is ineffective for generation of channelrhodopsin-mediatedcurrents or motility responses in flagellates, but is absorbed by thephotosynthetic pigments. The spectrum was corrected for photon density,taking into account the logarithmic fluence-response dependence (FIG.2). The spectral maximum at 531 nm was determined by Gaussian fit of themain peak (the solid line in FIG. 3). The 90-nm half-bandwidth of thespectrum is typical of retinal proteins, indicating that a singlerhodopsin species is responsible for the light-induced currents in M.viride. Significant light-sensitivity of the electric response isobserved even beyond 620 nm. This long-wavelength action spectrum andthe fast kinetics of the photoelectric current identified this organismas a candidate for source for cloning a channelrhodopsin that wouldfacilitate and improve optogenetics applications.

Example 5 Primary Structure M. viride Opsin

The opsin sequence cloned from M. viride (593 residues, peptide sequenceprovided as SEQ ID NO: 1) consists of a 7TM domain and an extendedC-terminal domain, as is characteristic of other knownchannelrhodopsins. The channelopsin is the apoprotein, whilechannelrhodopsin is the protein and retinal. Strictly speaking the aminoacid sequences (SEQ ID NOS:1-4) define the opsin, but this is also thesequence of the rhodopsin, which is the same. Most of the residues knownto form the retinal-binding pockets of other sensory rhodopsins and BRwere conserved. However, overall homology of this sequence to otherchannelrhodopsins is lower than between any two of them: its 7TM domainshows 38% identity and 52% similarity with ChR1, 35% and 51% with ChR2,36% and 51% with VChR1, and 37% and 53% with VChR2. The genomes of C.reinhardtii and V. carteri are completely sequenced, and each containsonly two channelopsin genes. Complete genomic information is notavailable for M. viride and there is a possibility that its genome alsocontains more than one channelopsin. Based on sequence homology the M.viride protein could not be distinguished as a channelrhodopsin 1 or 2since it is only slightly closer to the two channelrhodopsin 1 sequences(FIG. 4). Also this sequence lacks two structural features thatdifferentiate ChR1/VChR1 from ChR2/VChR2. First, there is no Glu residuecorresponding to E87 (ChR1 numbering), which is implicated bymutagenesis as required for the pH-dependent spectral shiftcharacteristic of ChR1/VChR1 (Tsunoda, S. P., and P. Hegemann. Glu 87 ofchannelrhodopsin-1 causes pH-dependent color tuning and fastphotocurrent inactivation. Photochem. Photobiol. 85:564-569, 2009). Alsothe position of Y226 (ChR1)/N187 (ChR2), conserved in VChR1 and VChR2,respectively, is one of the molecular determinants of spectralsensitivity, desensitization and kinetics, by which ChR1 differs fromChR2 (Wang, H., Y. Sugiyama, T. Hikima, E. Sugano, H. Tomita, T.Takahashi, T. Ishizuka, and H. Yawo. Molecular determinantsdifferentiating photocurrent properties of two channelrhodopsins fromChlamydomonas. J. Biol. Chem. 284:5685-5696, 2009). In the M. virideprotein this site is occupied by a Trp residue.

In the two known channelrhodopsin pairs, the more red-shifted one isdefined as channelrhodopsin 1. The spectral sensitivity of M. viridechannelrhodopsin is more red-shifted than that of any previously knownspecies, including the most red-shifted VChR1 (see below). Therefore, itwas named MChR1 (and not 2).

Unexpectedly, several residues in the 7TM region previously consideredas defining features of the channelrhodopsin family based on the fourknown homologs, were not conserved in MChR1, as described below underExample 7, below. The C-terminal domain of MvR1 shows even lowerhomology to that of other channelrhodopsins than the 7TM domain.

Example 6 Channel Activity in Mammalian Cells

The light-gated channel activity of Mesostigma rhodopsin was tested inHEK293 cells (FIG. 6). Characterization of the 7TM domain of MChR1 (331residues: SEQ ID NO: 1) and photocurrent generated by MChR1 undersustained illumination decayed from a peak to a plateau, a phenomenoncalled inactivation (FIG. 6). The amplitude and the sign of thephotocurrent depended on the holding potential with the reversalpotential close to zero (FIG. 7). The peak current and the plateau levelhad identical I-V curve shapes and reversal potentials. The relativelysmall inactivation of MChR1-generated currents showed it is functionallycloser to ChR1 than to ChR2, as the former exhibits a lower degree ofinactivation (16). The mean peak value of MChR1 photocurrents at thesaturating light intensity and −60 mV holding potential was 276±97 pA(n=12, mean±SEM), which is comparable to that reported for VChR1 inneurons, and to the measurements of VChR1 in HEK cells. The plateauamplitude of MChR1 photoresponses saturated at lower light intensitiesthan the peak value (FIG. 8) and the degree of inactivation increasedwith the light intensity. When successive pulses were applied with avariable dark interval, the amplitude of the peak recovered with (theta)τ ˜3.5 s at pH 7.4, which was faster than that reported in otherchannelrhodopsins. In addition to this short-term process, another,long-term recovery was made evident by the fact that the first lightstimulus always elicited a larger response than any subsequent one, evenafter minutes of dark adaptation.

Estimation of the absorption properties of a receptor responsible for aphotobiological response is usually done using action spectroscopy. Forthe action spectrum to faithfully reflect the absorption spectrum of thepigment, the response needs to be measured far from saturation, ideallyin the linear range of the intensity-response curve. Also, the actionspectrum should be measured in response to a short stimulus to avoidsecondary photochemistry, which may produce a spectrally-dependentmixture of intermediates, affecting the action spectrum. The initialslope of the photocurrent (assessed from the mean amplitude of thesignal recorded during the first 50 ms of illumination withlow-intensity light) was measured to evaluate spectral properties of theground state of MChR1 in HEK cells. This parameter showed a near lineardependence on light even at such intensities when both the peak andplateau levels saturated (FIG. 9) and hence could be easily correctedfor equal photon density.

The action spectra of MChR1 light-gated channel activity in HEK cellsmeasured as outlined above at three values of external pH are shown inFIG. 10. To reveal the position of their maxima, the main peaks of thespectra were fitted with a Gaussian function (as shown in FIG. 3 for thespectrum in native M. viride cells). At all three pH values, thespectral maximum was at 528 nm, which corresponded within 3-nm accuracyto the maximum of the spectral efficiency of the pigment in native M.viride cells (FIG. 3).

The long-wavelength spectral sensitivity found in MChR1 is one of theproperties highly desirable for neurobiological applications. The mostred-shifted channelrhodopsin variant previously reported is VChR1 fromV. carteri. The action spectra of light-gated channel activity of VChR1was characterized under the same experimental conditions used for MChR1and it was determined, using the same fitting algorithm, that the VChR1spectrum peaked at 520 nm at neutral pH (FIG. 11, solid line), which was˜8 nm less than that measured in MChR1 (FIG. 10). The 520 nm peak of theVChR1 spectrum corresponded to the absorption maximum of the isolatedpigment measured at this pH. Acidification of the external medium led tothe appearance of a red-shifted shoulder in the VChR1 action spectrum(FIG. 11, dashed line). The maximal absorption of purified VChR1 hasbeen reported to shift to 540 nm at low pH, indicating conversion of thepigment to a protonated form. These findings suggested that eitherpartial conversion occurred in HEK cells, or that the conductance of theprotonated state was much smaller than that of the 520 nm form.

The main difference between the two red-shifted channelrhodopsins wasthat the current decay after switching off the light was much faster inMChR1 than in VChR1. In both cases, the decay kinetics was better fitwith two exponentials. At neutral pH, the time constants of bothcomponents were respectively smaller in MChR1 than in VChR1 (FIG. 12).In addition, the relative amplitude of the slow component was smaller inMChR1, further increasing the rate of the overall decay. The fastcomponent of MChR1 was further characterized due to the smallcontribution of the slow component to the decay kinetics in MChR1. Itsrate strongly increased upon acidification of the external medium fromthe pH 9.0 to 5.3 (FIG. 13, filled symbols), whereas in VChR1 it wasonly slightly increased (FIG. 13, open symbols). However, the rate ofthe slow component in VChR1 showed a stronger dependence on pH than thefast component.

Example 7 Functional Analysis Using Mutagenesis

MChR1 showed some typical light-gated channel activity upon heterologousexpression but not all structural features found in otherchannelrhodopsins were conserved in its primary sequence. Tocharacterize the functional importance of these features, MChR1 pointmutants (V102E and A116E) were generated and compared to knownchannelrhodopsins and their activity analyzed.

V102E and A116E Mutants:

All previously identified channelrhodopsins contain an array of five Gluresidues in the predicted second helix, but only three of these areconserved in MChR1 (FIG. 5). Thus it was expected that introducing themissing glutamates (corresponding to E83 and E97 in the ChR2 sequence)in the MChR1 sequence would enhance its channel activity. However, bothmutations resulted in a substantial decrease in the whole-cell currentamplitude (˜7-fold in A116E and more than 100-fold in V102E). Thisamplitude depends on the properties of individual channelrhodopsinmolecule and on the number of functional molecules in the plasmamembrane. Based on visual observations, the intensity of EYFPfluorescence in the membrane was not significantly changed by the V102and A116E mutations. The presence of comparable EYFP fluorescencesuggests that the expression level was not affected, although it doesnot exclude that the protein was misfolded. These mutations alsoinhibited the channel function of the MChR1 molecule, as was revealed bya significant slowing down of the current kinetics (FIG. 14 for A116E;data for V102E are not shown). In the double mutant V102E/A116E (bothglutamates introduced) the photocurrents were below the detection limit.

A153/H/R Mutants:

All previously identified channelrhodopsins have a His residue at theposition of the proton donor in BR (D96), and its replacement with Argresults in an increase in the stationary current amplitude in ChR2 (10).When Ala found in MChR1 in this position was replaced with His or Arg,the current was completely suppressed without decreasing the EYFPfluorescence in the membrane.

C147A and D175A Mutants:

For previously identified channelrhodopsins, it has been suggested thatresidues C128 and D156 in ChR2 form a hydrogen-bond between helices Cand D that is important for channel gating and that its disruption leadsto a dramatic increase in the lifetime of the channel's open state. Thecorresponding residues (C147 and D175) are conserved in MChR1. Replacingeither of these residues with Ala led to a large decrease in the EYFPfluorescence indicating a decrease in protein expression levels. Currentamplitude was also significantly decreased to a maximal signal of ˜20pA. However, in agreement with the ChR2 result, analysis of the currentkinetics showed a similar dramatic increase in the decay time (FIG. 14for D175A; data for C147A are not shown).

Channelopsins Derived from Chlamydomonas Example 8 Source and Growth ofAlgae

Algal strains were obtained from the Culture Collection of Algae at theUniversity of Texas (UTEX), Austin, Tex. and the National Center forCulture of Marine Phytoplankton (CCMP), West Boothbay Harbor, Me.Chlamydomonas augustae (UTEX SNO134) and Chlamydomonas yellowstonensis(UTEX B SNO155) were grown in Bold 1NV medium (described by Starr, R. C.and J. A. Zeikus (1993) UTEX—the culture collection of algae at theUniversity of Texas at Austin. J. Phycol. 29 (suppl.), 1-106) underillumination of 14 μmol photon×M⁻²×s⁻¹ at 4° C. or 16° (C. augustae), or4° C. (C. yellowstonensis). Chlamydomonas raudensis (CCMP 1619) wasgrown in modified Bold's basal medium (described by Bischoff, H. W. andH. C. Bold (1963) Phycological studies IV. Some soil algae fromEnchanted Rock and related algal species. University of TexasPublication 6318, 1-95) containing vitamins and three times the amountof nitrogen and vitamins as described at the website of the CultureCollection of Algae and Protozoa (UK) under illumination of 5 μmolphoton×m²×s⁻¹ at 4° C. Illumination was set to a 16 h light, 8 h darkcycle and was provided by cool-white fluorescent lamps.

Example 9 Cloning and Expression of Chlamydomonas Channelrhodopsins

Homology Cloning of Sequences.

Algae were inoculated from plates into 25 mL liquid medium in 250 mLflasks and grown for 18 hr at 16° C. (C. yellowstonensis) or 4° C. (C.raudensis). Total RNA was extracted with Trizol reagent (Invitrogen,Carlsbad, Calif.). First strand cDNA was synthesized with theTranscriptor first strand cDNA synthesis kit (Roche Diagnostics,Mannheim, Germany) using oligo-d(T) primer. The degenerate primers weredesigned according to the conserved regions of the four earlier knownchannelopsins from C. reinhardtii and V. carteri, and their degeneracywas reduced by including only the most frequently used codons. Theforward primer was SYHB-F (5′-TGC GGN TGG GAG GAG RTN TA-3′ (SEQ ID NO:13), and the reverse primers were SYOG-R (5′ AGR ATR TGC TCR TGRATC-3′(SEQ ID NO: 14)) for C. augustae and C. yellowstonensis, andSYLPEF-R (5′-RCC CTT SGG NAC SGT RTG-3′ (SEQ ID NO: 15)) for C.raudensis. The PCR (polymerase chain reaction) program was as follows:denaturation at 98° C. for 2 min, followed by 30 cycles of 98° C. for 30s, 49° C. for 45 s, 72° C. for 55 s, and final extension at 72° C. for 5min. PCR fragments were cloned into the pGEM-T Easy vector (Promega,Madison, Wis.) and sequenced. For the fragments that showed homologywith channelopsins, 3′ and 5′ RACE (rapid amplification of cDNA ends)was performed using the SMARTer RACE cDNA amplification kit (ClontechLaboratories, Mountain View, Calif.). Overlapping RACE fragments werejoined by fusion PCR to obtain full-length cDNA, which was cloned intothe pGEM-T Easy vector and sequenced. Fragments encoding for the 7TMdomains (residues 1-352 for the C. augustae and C. raudensis sequences,and 1-354 for C. yellowstonensis) were inserted between BamHI and NotIsites to replace the VChR1 sequence in the pcDNA3.1/VChR1-EYFP mammalianexpression vector provided by K. Deisseroth (Stanford University). Thevector map and sequence are available at the Optogenetics ResourceCenter website. The presence of a fluorescent tag is not expected toaffect channelrhodopsin properties, as has been shown by quantitativecomparison of photocurrents generated by YFP-, mCherry- and myc-taggedChR2.

Example 10 Structural Sequence Analysis

Channelopsin sequences from C. augustae, C. yellowstonensis and C.raudensis. Channelopsin homologs were cloned from each of C. augustae(SEQ ID NO: 2, 715 amino acid residues; nucleotide Acc. No. JN596951),C. yellowstonensis (SEQ ID NO: 3, 717 residues, Acc. No. JN596948) andC. raudensis (SEQ ID NO: 4, 635 residues, Acc. No. JN596949). The newproteins consist of a predicted 7TM (rhodopsin) domain responsible forlight-gated channel activity and a C-terminal domain. ChR1/VChR1 andChR2/VChR2 form two distinct branches on the phylogenetic tree of their7TM domains (FIG. 17). The 7TM domains of the new Chlamydomonassequences do not show closer homology with either the ChR1/VChR1 branchor ChR2/VChR2 branch, when their overall sequence homology is concerned(FIG. 17). The 7TM domains from C. augustae and C. yellowstonensis arehowever very close to each other.

Two molecular determinants are conserved in ChR1/VChR1 and ChR2/VChR2sequences, respectively, and shown to determine their differentproperties: 1) Glu87 (ChR1 numbering) in the predicted first helix isresponsible for pH-dependent color tuning and fast channel inactivationof ChR1, as compared to ChR2; 2) Tyr226 (ChR1)/Asn187 (ChR2) in thepredicted fifth helix confers differences in spectral sensitivity,inactivation and kinetics between ChR1 and ChR2. According to thesecriteria, the sequences identified in C. augustae and C. yellowstonensisbelong to the ChR1/VChR1 class (FIG. 19). This placement is suggested bytheir red-shifted spectra (see below), characteristic of the ChR1/VChR1class, as compared to the ChR2/VChR2 class. In contrast, the sequencefrom C. raudensis belongs to the ChR2/VChR2 class according to the twoabove-mentioned molecular determinants.

To distinguish the new Chlamydomonas channelopsins from the previouslyknown ones from C. reinhardtii, species-specific abbreviations, i.e.,CaChR1 for the C. augustae sequence, CyChR1 for that from C.yellowstonensis, and CrChR2 for the C. raudensis sequence. The originalChR1 and ChR2 are referred to as such, without modification for thesource organism, because these sequences are already well-known underthese names.

In the 7TM domain, residues at the active sites characteristic of otherknown channelopsins are conserved in all three new homologs. Theseinclude: 1) Glu in the position of the Schiff base proton acceptor(Asp85 according to bacteriorhodopsin (BR) numbering); 2) His in theposition of the Schiff base proton donor (Asp96 in BR); 3) five Gluresidues in or near the predicted second helix; and 4) Cys128 and Asp156(ChR2 numbering) that form a predicted hydrogen bond between the thirdand fourth helices. Out of other residues known to form theretinal-binding pocket in BR, Tyr57, Gly122, Trp182, Asp212 and Lys216(BR numbering) are conserved in new channelopsins, as they are in allpreviously known ones. Positions of Tyr185 and Trp189 (BR numbering) areoccupied by Phe residues, as in all previously known channelopsins.

Phylogeny of the C-terminal domain does not match that of the 7TMdomain, although CaChR1 and CyChR1 again show close similarity (FIG.18). No helices are predicted in the C-terminal domains of either of thenew channelopsin sequences, in contrast to ChR1. As in other previouslyknown channelrhodopsins, the C-terminal domains of the new channelopsinscontain several highly conserved regions with no homology to any otherso far known protein interspersed with repeats that vary in length andamino acid composition among different channelopsin variants. Longstretches of Gly-Met repeats and Met-rich regions are found in CaChR1and CyChR1, and Gln repeats are found in all three new channelopsins.Such repeats, known as homopolymeric tracts, occur in many eukaryoticproteins and have been associated with protein-protein orprotein-membrane interactions. A highly conserved region of about 40residues at the very end of the C-terminal domains present in all so farknown channelrhodopsins but MChR1 shows homology to domains infibrinogen and ABC transporters that are responsible for proteinmultimerization and protein-protein interaction. In algal cellschannelrhodopsins are confined to the membrane area above the eyespotand are associated with acetylated microtubules of the daughterfour-membered flagellar rootlet.

The extracellular N-terminal regions of CaChR1 and CyChR1 contain apredicted conserved N-glycosylation site. Such sites, although atdifferent positions, are also predicted in the N-termini of ChR1 andseveral other channelopsins, but not in CrChR2 (FIG. 19). Another suchsite conserved is located at the cytoplasmic end of the third predictedtransmembrane helix. While not being bound by such theory, there may bea possible requirement for glycosylation for correct folding andtargeting of channelrhodopsins may explain why no functionalchannelrhodopsin has been produced by expression in E. coli, despitemany attempts. All three new channelopsin sequences lack an additionalα-helix predicted in the N-terminus of ChR1 as a signal peptide, as doChR2 and both VChR1 and VChR2. However, such helix is predicted in thesequence from Haematococcus pluvialis.

In ChR1 and ChR2 from C. reinhardtii, three and one phosphorylatedresidues, respectively, were identified by phosphoproteomics of theeyespot fractions. These residues are found in the cytoplasmic loop nextto the 7TM domain that is highly conserved in all so far knownchannelopsin sequences, with the exception of MChR1. Out of the threephosphorylated residues of ChR1, Ser359 is conserved in all fiveChlamydomonas channelopsins (FIG. 19), and the corresponding residue(Ser321) is the only phosphorylated site detected in ChR2. Thr374 isunique for ChR1 (with Val found at this site in other sequences), andSer377 is conserved in four sequences with the conservative substitutionin CrChR2 (FIG. 19).

Proteins from psychrophilic organisms show characteristic biases inamino acid composition, compared to their meso- and thermophilichomologs, that are believed to increase flexibility at low temperatures.Among these are decreased percentages of Pro, Arg and Ala residues, andan increased percentage of Ile residues. The same trends are observed inChlamydomonas channelopsins: the combined percentages of Pro, Arg andAla residues in the sequences of ChR1, CaChR1 and CyChR1 are 20%, 15.7%and 16%, respectively, whereas the percentages of Ile are 4.9%, 5.9% and5.2%, respectively.

Example 11 Functional Characterization of Chlaymydamonas Channelopsins

HEK293 cells were transfected using the TransPass COS/293 transfectionreagent (New England Biolabs, Ipswich, Mass.). All-trans-retinal (Sigma)was added as a stock solution in ethanol at the final concentration of2.5 μM, unless otherwise indicated. Measurements were performed 48-72 hafter transfection with an Axopatch 200B amplifier (Molecular Devices,Union City, Calif.). The signals were digitized with a Digidata 1440Ausing pClamp 10 software (both from Molecular Devices). Patch pipetteswith resistances 2-5 MΩ were fabricated from borosilicate glass andfilled with the following solution (in mM): KCl 126, MgSO₄ 2, CaCl₂ 0.5,EGTA 5, HEPES 25, pH 7.2. The bath solution contained (in mM): NaCl 150,CaCl₂ 1.8, KCl 4, MgCl₂ 1, glucose 5, HEPES 10, pH 7.4, unless otherwiseindicated. For experiments at pH 9, Tris was used in the bath solutioninstead of HEPES. Unless otherwise indicated, the holding potential was−60 mV. Light excitation was provided by a Polychrome IV light source(T.I.L.L. Photonics GMBH, Grafelfing, Germany) pulsed with a mechanicalshutter (Uniblitz Model LS6, Vincent Associates, Rochester, N.Y.;half-opening time 0.5 ms). The light intensity was attenuated with thebuilt-in Polychrome system or with neutral density filters. Maximalquantum density at the focal plane of the 40× objective lens was ˜2×10²²photons×m⁻².

Absorption spectroscopy. Absorption spectrum of partially purifiedCaChR1 in the UV—visible range was recorded on a Cary 4000spectrophotometer (Varian, Palo Alto, Calif.).

The 7TM domains of all three Chlaymydamonas channelopsins showed robustexpression in the plasma membrane of HEK293 cells, as indicated byfluorescence of their EYFP-tags. CaChR1 and CyChR1 exhibited light-gatedchannel activity in this system, but no currents could be recorded uponexpression of CrChR2. The kinetics of the currents generated by CaChR1and CyChR1 were however quite different from that generated by ChR1.FIG. 20 shows typical signals recorded at the maximal light intensityunder our standard conditions, i.e., bath pH 7.4, and holding potential(V_(hold)) −60 mV. Upon switching on the light, currents generated byChR1 underwent a rapid initial rise with a time constant (τ) ˜1 ms,reached a peak and rapidly (τ˜5 ms) decreased under sustainedillumination, i.e., inactivated to a lower level (FIG. 20, black line).In many cells the currents showed a subsequent slight increase withτ˜200 ms. This behavior closely resembled the results reported for ChR1.In contrast, the rise of CaChR1- and CyChR1-generated currents wasbiphasic. The first rapid phase was similar to that of ChR1-generatedcurrents, but it was followed by a slower rising phase with τ˜20 ms(FIG. 20). The relative contributions of these two components variedfrom cell to cell. After reaching a peak, CaChR1- and CyChR1-generatedcurrents exhibited very slow inactivation (τ˜500 ms).

For all three new Chlaymydamonas channelrhodopsin variants the responseto the first flash showed a larger peak relative to the plateau level(measured at the end of the light pulse), which was not fully recoveredeven after 30 min dark interval, suggesting a contribution of a veryslow adaptation process, or irreversible bleaching of an unstablefraction of the pigment. However, no difference was observed betweenresponses to the second and all subsequent flashes recorded with 30 sdark intervals. Under these conditions, the peak to plateau ratio at themaximal light intensity was 1.7±0.2 (mean±SEM, n=8) for ChR1, close tothe earlier reported results. For both CaChR1 and CyChR1 this ratio wassignificantly smaller: 1.2±0.1 (mean±SEM, n=12 and n=6, respectively).The absolute plateau amplitude was 101±25 pA (mean±SEM, n=8) for ChR1,64±9 pA (mean±SEM, n=12) for CaChR1, and 49±13 pA (mean±SEM, n=6) forCyChR1. After switching off the light, the currents decayedbiexponentially with τ˜15 and ˜120 ms for CaChR1, ˜13 and ˜150 ms forCyChR1, which was slower than ˜4 and ˜18 ms measured for ChR1 (FIG. 21),but close to that for ChR2.

The most widely used channelrhodopsin variant, ChR2, generates largeenough currents in HEK cells even without the addition of exogenousretinal, indicating that its trace amount present in these cells issufficient for reconstitution of functional protein. However, currentsin cells transfected with the new channelrhodopsins or ChR1 wereconsiderably smaller if no exogenous retinal was added: their plateaulevels were only ˜8% for CaChR1 and ˜26% for ChR1 (data for CyChR1 arenot shown), relative to the results obtained with the respectivechannelrhodopsins in the presence of 2.5 μM exogenous retinal.

As described previously for ChR1-generated currents, the dependence ofthe plateau amplitude on the stimulus intensity saturated earlier thanthat of the peak (FIG. 22). In fact, the curve for the peak amplitudewas biphasic and showed two levels of saturation, the first of whichcorresponded to that of the plateau level, whereas the second was atmore than 10-fold higher light intensity. Therefore, the magnitude oflight inactivation, calculated as the difference between the peak andplateau amplitudes relative to the peak amplitude, increased with lightintensity and did not saturate even at the highest available intensities(FIG. 24, squares). In contrast, the curves for both peak and plateauamplitudes of CaChR1-generated currents consisted of two phases (FIG.23), so that the magnitude of light inactivation reached the maximum at10% maximal light intensity and then declined (FIG. 24, circles).Similar behavior was observed for CyChR1-generated currents (data notshown).

CaChR1- and CyChR1-generated currents showed a typical dependence on theholding potential (V_(hold)) (FIGS. 25, 26 and 27). The reversalpotentials (V_(r)) were similar to that for ChR1 and close to zero underour experimental conditions.

CaChR1 and CyChR1 were tested for proton permeability by measuringcurrent-voltage relationships (I-V curves) under variable external pH todetermine if they were highly proton-selective channels. Acidificationof the external medium caused an increase in the current amplitude at agiven voltage and a shift of the reversal potential to more positivevalues. The magnitude of this shift for CaChR1 and CyChR1 was similar tothat for ChR1 (FIGS. 25, 26 and 27). Therefore, it can be concluded thatboth CaChR1 and CyChR1 are mostly selective for protons.

The rate of current decay after switching off the light increasedslightly for both, CaChR1 and CyChR1 upon a change of the bath pH from7.4 to 5.4 (FIGS. 29 and 30). This contrasts with reports for ChR1 thatthe rate of current decay after switching off the light decreases atacidic bath pH.

The spectral sensitivity of photocurrents was analyzed under lowintensity light, as described in EXAMPLE 4 above. The spectral maximafor the new Chlamydomonas channelrhodopsins, CaChR1 and CyChR1, were at520 nm at pH 7.4 (FIG. 32 and 32 black squares), which is 40 nm longerthan that of the action spectrum of ChR1-generated currents in oocytes,and the absorption spectrum of purified ChR1 at neutral pH. To rule outa possible influence of a different expression system and/or a differentalgorithm of construction of the spectra, the action spectrum ofcurrents generated by ChR1 in HEK cells was also measured. It showed amaximum at ˜480 nm at pH 7.4 (FIG. 31, open triangles), but had a broadshape with significant absorption above 500 nm, indicating acontribution of the red-shifted deprotonated form of the pigment as wasreported in oocytes. For both CaChR1 and CyChr1, the spectra measured atneutral (7.4) and acidic (5.4) pH were identical (FIGS. 31 and 32), incontrast to the spectrum of ChR1, which showed a significant red shiftupon acidification of the medium. However, a small ˜10 nm blue shift wasobserved upon the pH change from 7.4 to 9 for both CaChR1 and CyChR1(FIGS. 31 and 32). The maximum of the action spectrum of photocurrentsgenerated by ChR1 in native C. reinhardtii cells is 505 nm, whereas thatof currents generated by ChR1 in heterologous systems at neutral pH isblue-shifted by at least 25 nm (FIG. 31). Expression of CaChR1 in P.pastoris in the presence of all-trans-retinal yielded photoactivepigment. The absorption spectrum of CaChR1 purified from Pichia exactlymatched the action spectrum of photocurrents generated by this pigmentin HEK cells, which indicated that its native state was essentiallypreserved in detergent (FIG. 33).

Example 12 Cloning and Expression in Yeast

Expression and purification from Pichia. The 7TM domain of CaChR1 (1-352residues) was cloned into the pPIC9K vector (Invitrogen, Carlsbad,Calif.) between EcoRI and NotI sites in frame with two TEV proteasesites at the N-terminus and before a C-terminal 6His-tag. The resultantplasmid was linearized with BspEI and transformed into the P. pastorisSMD1168 (his4, pep4) strain by electroporation according to themanufacturer's instructions. First, transformants were screened forplasmid integration by their ability to grow on hystidine-deficientplates, and second, for multiple inserts by their geneticin resistance.A single colony that grew on 4 mg/mL geneticin was selected. A starterculture was inoculated in 500 mL BMMY (buffered minimal methanol yeast)medium. Expression was induced by the addition of 0.5% methanol every 24h in the presence of 30 μM all-trans-retinal. Cells were grown for twodays, harvested by low-speed centrifugation and disrupted by a beadbeater. Membrane fragments were collected by centrifugation for 1 h at48,000 rpm and solubilized by incubation with 2% dodecyl maltoside for 1h. The protein was purified on a Ni-NTA agarose column (Qiagen, Hilden,Germany). The protein yield was 6.4 mg/L of culture, as estimated fromabsorbance at 520 nm.

Enhancement of Long-Wavelength Sensitivity of Optogenetic MicrobialRhodopsins Example 13-A1 Archaerhodopsin from Halorubrumsodomense (AR-3)

To determine the effect of A2 retinal on the proton pump AR-3 expressedin E. coli cells, since this expression system allows quantitativemeasurements of absorption and fast charge movements within rhodopsinmolecules. To do this, AR-3 was expressed in the presence of A1 or A2retinal. The AR-3 coding sequence was received from Dr. E. S. Boyden(Massachusetts Institute of Technology, Boston, Mass.) and expressed inE. coli cells as described earlier for other microbial rhodopsins (seeJung, K.-H., Trivedi, V. D., and Spudich, J. L. Demonstration of asensory rhodopsin in eubacteria, Mol. Microbiol. 47, 1513-1522, 2003 andWang, W.-W., Sineshchekov, O. A., Spudich, E. N., and Spudich, J. L.Spectroscopic and photochemical characterization of a deep oceanproteorhodopsin, J. Biol. Chem. 278, 33985-33991, 2003) in the presenceof 5 ul of all-trans A1 retinal (Sigma), or all-trans A2 retinal (atleast 99% pure as tested by HPLC; a gift from Dr. R. K. Crouch, MedicalUniversity of South Carolina, Charleston, S.C.—but also available from,for example, Toronto Research Chemicals, Ontario, Canada). Cells werewashed in distilled water and transferred to low-ionic strength medium(in mM): NaCl 1.5, CaCl₂ 0.15, MgCl₂ 0.15, Tris 5, pH 7.2. Absorptionspectra were recorded on a Cary 4000 spectrophotometer with integratingsphere (Varian, Palo Alto, Calif.). Absorption spectrum of cells withoutinduction of expression was subtracted from those expressing A1- orA2-reconstituted AR-3 to correct for scattering and intrinsic proteinabsorption. Photocurrents in suspension of the cells were generated by 8ns laser flash applied along the direction between two platinumelectrodes (as described in Sineshchekov, O., and Spudich, J.Light-induced intramolecular charge movements in microbial rhodopsins inintact E. coli cells, Photochem. Photobiol. Sci. 3, 548-554, 2004).

The absorption spectra, corrected for light scattering and minordifferences in the amount of cytochromes caused by expression of aforeign protein are presented in FIG. 34. The absorption maximum of theA2-reconstituted pigment was 35-nm red-shifted from that of theA1-reconstituted AR-3 (558 nm to 593 nm). The shift of the wavelength ofhalf-maximal absorption on the red slope of the spectra was evengreater, 40 nm (from 599 to 639 nm). This may indicate the expectedwidening of the band with its shift to longer wavelengths, as has beennoted previously in animal visual pigments reconstituted with A2 retinal(Bridges, C. D. Spectroscopic properties of porphyropsins, Vision Res.7, 349-369, 1967). A poorly resolved shoulder was observed on theshort-wavelength slope of the spectrum of the A2-reconstituted pigment.This shoulder may indicate a very small amount of A1 retinalcontamination. However, its position was at a shorter wavelength thanthe maximum of the A1-reconstituted spectrum.

The pigments reconstituted with A1 and A2 retinals demonstrated verysimilar kinetics of intramolecular charge movements with a slightlyfaster decay of the fast current associated with proton transfer to theacceptor, which corresponds to formation of the M intermediate (thecurrent traces in FIG. 35), and slightly slower reprotonation of theSchiff base (better resolved in the charge traces in FIG. 35). Theabsolute amplitude of the fast photocurrent was ˜3-fold lower in thesample incubated with A2 retinal, which correlates roughly with the2-fold lower expression and faster decay kinetics. Thus, the absolutequantum yields of proton transport in the A1 and A2 forms appear to becomparable.

The spectral sensitivity of photoelectric responses in E. coli cells aswell as in HEK293 cells was measured at very low light intensities (inthe range where the dependence was close to linear) to avoid distortionand facilitate correction for the number of photons.

Whole-cell patch clamp recording in HEK293 cells: HEK293 (humanembryonic kidney) cells were transfected using the TransPass COS/293transfection reagent (New England Biolabs, Ipswich, Mass.). A1all-trans-retinal (Sigma) was added as a stock solution in ethanol atthe final concentration of 2.5 μM. A2 all-trans-3,4-dehydroretinal (>99%pure as tested by HPLC; a gift from Dr. R. K. Crouch, Medical Universityof South Carolina, Charleston, S.C.) was added at a final concentrationof 5 μM. Measurements were performed 48-72 h after transfection with anAxopatch 200B amplifier (Molecular Devices, Union City, Calif.). Thesignals were digitized with a Digidata 1440A using pClamp 10 software(both from Molecular Devices). Patch pipettes with resistances of 2-5 MΩwere fabricated from borosilicate glass and filled with the followingsolution (in mM): KCl 126, MgCl₂ 2, CaCl₂ 0.5, EGTA 5, HEPES 25, pH 7.4.The bath solution contained (in mM): NaCl 150, CaCl₂ 1.8, MgCl₂ 1,glucose 5, HEPES 10, pH 7.4. The holding potential was −60 mV. Lightexcitation was provided by a Polychrome IV light source (T.I.L.L.Photonics GMBH, Grafelfing, Germany) pulsed with a mechanical shutter(Uniblitz Model LS6, Vincent Associates, Rochester, N.Y.; half-openingtime 0.5 ms). The light intensity was attenuated with the built-inPolychrome system or with neutral density filters. Maximal quantumdensity at the focal plane of the 40× objective lens was ˜2×10²²photons×m⁻².

In the case of continuous light excitation of HEK293 cells only theinitial part of the current signals up to 20 ms was measured to minimizethe involvement of possible photoreactions of photocycle intermediates.Photocurrents were normalized according to the number of photons in eachlaser flash or light pulse.

In full agreement with the difference in the absorption spectra, theaction spectrum of the charge movement in A2-reconstituted AR-3 isred-shifted by ˜35 nm with the half-maximum efficiency at >640 nm (FIG.36, solid symbols, solid lines). This confirms that the charge movementregistered in a suspension of E. coli cells is generated by theA2-reconstituted fraction of AR-3, and not by the fraction reconstitutedwith trace amounts of A1 retinal. A similar shoulder at ˜540 nm asobserved in the absorption spectrum is apparent in the action spectrum.

A1- and A2-reconstituted AR-3 were compared expressed in HEK293 cells.An excess amount (5 uM) of A2 retinal was added, because HEK cells areknown to contain endogenous A1 retinal). Light-induced hyperpolarizingcurrents of A2-reconstituted AR-3 did not significantly differ in theamplitudes and kinetics of corresponding currents generated by AR-3reconstituted with A1 retinal (data not shown). When the cells wereincubated with A1 or A2 retinal for two days, the spectra for thephotocurrents in HEK cells were essentially identical to the actionspectra of charge movement measured in E. coli cells (FIG. 36, opensymbols, dashed lines). However, on the third day of incubation ofHEK293 cells with A2-retinal the maximum of the action spectra shiftedto shorter wavelengths and a clear band corresponding to the maximum ofthe action spectrum of A1-reconstituted AR-3 appeared (FIG. 37, solidsymbols, thick solid line). An increased relative contribution of the A1pigment as compared to the A2 pigment upon an increase in the incubationtime is obvious from the difference spectra (thin solid line in FIG.37).

The increase in the amount of AR-3 reconstituted with endogenous A1between day 2 and day 3 is most probably due to activation of itssynthesis by the excess of A2 retinal. A similar, but even morepronounced effect was observed in channelrhodopsins.

Example 14 Channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2)

CrChR2, which activates neuron firing, is the most widely usedoptogenetic tool because of its high ion conductance and/or expressionlevel in animal cells. The major disadvantage of CrChR2 is itsshort-wavelength absorption with a long-wavelength half-maximalefficiency at ˜500 nm (squares in FIG. 38). Incubation of HEK293 cellstransfected with CrChR2 with A2 retinal caused significant changes inthe action spectrum of light-induced currents (circles in FIG. 38). Theoverall shape of the spectrum clearly indicates contribution of twopigment species in current generation. The strongly pronounced shoulderabove 500 nm may be due to A2-reconstituted CrChR2, whereas thestructured short-wavelength slope reflects a significant contribution ofA1-reconstituted CrChR2. This contribution is stronger than in the caseof AR-3, and contrary to the pump appears already after 2 days ofincubation of HEK cells with A2 retinal. This more rapid appearancesuggests a greater difference between affinities to A1 and A2 retinal inCrChR2, as compared to AR-3.

To characterize the absorption properties of A2-reconstituted CrChR2,the action spectrum of pure A1-reconstituted CrChR2 (squares in FIG. 38)was subtracted and multiplied by different coefficients, from the actionspectrum obtained after incubation with A2-retinal (circles). The mostspectrally reasonable difference curve (dashed line in FIG. 38) wasobtained assuming that the photoelectric response sensitized by CrChR2reconstituted with endogenous A1 comprises about 30% of the combinedaction of the A1- and A2-reconstituted pigments. The deduced spectrum ofpure A2-reconstituted ChR2 was wider than that of the A1-reconstitutedform, as would be predicted from its longer absorption. It lacked thefine structure evident in the A1 pigment and had a maximum at ˜508 nm,i.e. at 35 nm longer wavelength than the A1-reconstituted pigment.

Despite the significant contribution of the A1 retinal-reconstitutedform of CrChR2, the addition of A2 retinal produces a red shift inspectral sensitivity (assessed by the wavelength of half-maximalresponse) by >30 nm. However, the A1 and A2-reconstituted CrChR2 appearto be comparable since photoelectric currents induced by green lightabsorbed only by the A2 form reach similar high values (above 1 nA, FIG.39A) as the A1 pigment. Correct comparison of the kinetics of thephotocurrents generated by A1- and A2-reconstituted CrChR2 can only bedone at low light intensities to avoid saturation effects in the systemof two pigments with overlapping absorption. As shown in FIG. 39B,photocurrents generated in response to 440 nm light (absorbed primarilyby the A1 form), and to 530 nm light (absorbed essentially only by theA2 form) are almost identical. In summary, substitution of A1 retinalwith A2 retinal does not significantly affect the channel properties ofCrChR2, but shifts its absorption by 35 nm to the red.

Channelrhodopsin-1 from Chlamydomonas reinhardtii (CrChR1): Another C.reinhardtii channelrhodopsin, CrChR1, mediates phototaxis in nativealgal cells with a maximum at 505 nm. However, upon expression in animalcells the absorption spectra of CrChR1 at neutral pH peaks at a muchshorter wavelength (485 nm at pH 7.4 under our experimental conditions).Incubation of HEK cells transfected with CrChR1 with A2 retinal shiftedthe main maximum of the action spectrum by ˜30 nm to 515 nm.Simultaneously, a strongly pronounced shoulder at ˜555 nm appeared inthe action spectrum. This shoulder can be interpreted as the appearanceof the protonated form of CrChR1, which with A1 retinal was observedonly at low pH and had a maximum at 500-505 nm. Thus, substitution of A1retinal by A2 retinal not only shifted the spectral maxima of both thedeprotonated and protonated forms of CrChR1, but also shifted the pK_(a)of the Schiff base counterion to higher values. A similar effect of A2on the pKa of a color transition has been reported earlier inbacteriorhodopsin. As a combined result of these two effects, thewavelength of half-maximal efficiency of the long-wavelength of CrChR1was shifted upon incubation with A2 retinal by 45 nm from that of the A1retinal-reconstituted pigment. The dashed line in FIG. 40 illustratesthe deduced absorption spectrum of A2-reconstituted CrChR1 assuming thatthe photoelectric response sensitized by A1-reconstituted CrChR1comprises about 30% of the combined action of A1- and A2-reconstitutedpigments.

New Long-Wavelength Channelrhodopsins from Chlamydomonas augustae(CaChR1) and Mesostigma viride (MvChR1):

CaChR1 is a channerhodopsin variant recently identified in thepsychrophilic species C. augustae. In contrast to CrChR1, its maximalabsorption at neutral pH is at 520 nm. MvChR1 from M. viride is to datethe most red-shifted native channelrhodopsin, with a peak sensitivity atneutral pH at ˜530 nm. The addition of A2 retinal to HEK cellstransfected with CaChR1 or MvChR1 led to the appearance of stronglyred-shifted pigment forms obvious from the shape of the action spectra(FIGS. 41 and 42). The long-wavelength slope of the CaChR1 spectrum wasshifted to 589 nm at the level of 50% efficiency, more than 20 nm fromthat measured with A1 retinal, whereas the position of the maximumshifted to a smaller degree. In the case of MvChR1, reconstitution withA2 retinal shifted the long-wavelength slope of the spectrum by >40 nm,as compared to that measured upon reconstitution with A1 retinal.

In some embodiments, the long-wavelength sensitivity of optogeneticmicrobial rhodopsins is enhanced using 3,4-Dehydroretinal (A2 retinal).As described herein, the proton pump AR-3 and four testedchannelrhodopsin variants, CrChR1, CrChR2, CaChR1 and MvChR1,incorporated A2 retinal and produced functional proteins, the spectralsensitivity of which was significantly red-shifted from those of thecorresponding A1 retinal pigments. Such spectral shifts are expected tobe beneficial for optogenetic applications, especially in live animals,because light scattering decreases with the increase of wavelength.However, light scattering is not the only factor to consider; another,especially significant in brain tissue studies, is absorption byhemoglobin. Out of all tested channelrhodopsins, only CaChR1 and MvChR1reconstituted with A2 retinal showed significant sensitivity towavelengths above the long-wavelength boundary of hemoglobin absorption(FIG. 43).

To estimate the potential benefits of the use of A2 retinal inneuroscience optogenetic applications, the total number of actinicphotons absorbed by the corresponding A2 and A1 pigments over thevisible spectral range at different depths of brain tissue wascalculated. The action spectra of photocurrents recorded in HEK293 cellsthat express the corresponding opsins incubated with A1 or A2 retinalwere multiplied by the spectral distribution of light intensitiesderived from absolute values of light attenuation by brain tissue. Thearea under resultant curves proportional to the number of photonsabsorbed by each pigment was plotted in FIG. 44 as a function of thedistance from the brain surface. The curves were normalized to thevalues at the surface to compare attenuation of actinic light fordifferent pigments.

Based on this calculation the total absorption of short-wavelengthchannelrhodopsins (CrChR2 and CrChR1 deprotonated at neutral pH) willdecrease sharply within the tissue. Moreover, substitution of A2 for A1in CrChR2 will not improve significantly the situation, because adecrease in light scattering is compromised by an increase in hemoglobinabsorption. In contrast, substitution of A2 for A1 in CrChR1significantly increases the penetration depth of actinic light. However,this improvement is mostly due to a contribution of the protonated formof CrChR1, which in A2-reconstituted pigment appears at the neutral pHand has an absorption maximum above 550 nm (FIG. 40), i.e., ˜45 nmred-shifted as compared to the protonated form of A1-reconstitutedCrChR1 observed at low pH.

According to calculations, all tested rhodopsins with red-shiftedabsorption (CaChR1, MvChR1, and AR-3) are expected to permit optogeneticactivation in deep layers, even when bound to A1 retinal. Substitutionof A2 for A1 retinal in these rhodopsins will increase their efficiencysignificantly (FIG. 44). The greatest calculated effect was for MvChR1.At 1 cm depth the total number of photons absorbed by A2-bound pigmentwill be 13-fold greater than that absorbed by A1-bound pigment.

These measurements demonstrate that A2 retinal can be efficiently usedto improve the performance of optogenetic tools in cultured cells, andthe calculations above show that the benefits will be even greater forintact tissues. Therefore, in some embodiments, A2 retinal may be usedin living animals, it has been demonstrated that when vitamin A-deprivedrats received intraperitoneal injection of a retinal analog there wasrapid incorporation of the synthetic retinoid into a major fraction ofavailable opsin. This is the first instance of the use of retinal A toshift the absorption spectra of ontogenetic analogs.

The action spectra of photocurrents measured in HEK293 cells incubatedwith A2 retinal showed a rather prominent contribution ofA1-reconstituted pigments. This contribution is unlikely to be due toincorporation of trace amounts of A1 retinal in our A2 retinal stock,because: (i) no obvious A1-derived band appeared in A2-reconstitutedAR-3 produced in E. coli cells (FIGS. 35 and 36); (ii) the contributionof the A1-derived form in HEK293 cells increased with time (FIG. 37).The latter observation also cannot be explained by incorporation ofendogenous A1 retinal present in HEK293 cells, as only very smallcurrents could be recorded in control experiments in these cells withoutthe addition of retinoids. This suggests that in HEK cells exogenous A2retinal is partially converted to A1 retinal or activates biosynthesisof A1 retinal. It is noteworthy that the amplitudes of spectral shiftsupon substitution of A1 with A2 retinal reveal a significant differencebetween channelrhodopsins and other microbial rhodopsins. The magnitudeof the spectral shift in AR-3 is very close to the shifts measuredearlier in other long-wavelength microbial rhodopsins (<1,000 cm⁻¹). Incontrast, spectral shifts of all channelrhodopsins (or their protonatedforms, as in the case of CrChR1) are significantly larger, in the rangebetween ˜1,300 and ˜1,800 cm⁻¹ (FIG. 45). These shifts are also largerthan the shifts measured in animal visual pigments that exist in bothA1- and A2-bound forms in vivo, indicating structural differences in theretinal binding pockets in channelrhodopsins compared to other microbialrhodopsins.

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Without further elaboration, it is believed that one skilled in the artcan, using the description herein, utilize the present methods to itsfullest extent. The embodiments described herein are to be construed asillustrative and not as constraining the remainder of the disclosure inany way whatsoever. While preferred embodiments have been shown anddescribed, many variations and modifications thereof can be made by oneskilled in the art without departing from the spirit and teachings ofthe presently disclosed methods. Accordingly, the scope of protection isnot limited by the description set out above, but is only limited by theclaims, including all equivalents of the subject matter of the claims.The disclosures of all patents, patent applications and publicationscited herein are hereby incorporated herein by reference, to the extentthat they are consistent with the present disclosure set forth herein.

What is claimed is:
 1. An cDNA-derived nucleic acid comprising a nucleicacid sequence that encodes an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO:
 10. 2. The cDNA-derivednucleic acid of claim 1, wherein the polynucleotide encodes a rhodopsindomain.
 3. The cDNA-derived nucleic acid of claim 1, wherein the nucleicacid encodes the amino acid sequence of SEQ ID NO:
 2. 4. An expressionvector comprising a nucleic acid of claim
 1. 5. A host cell comprisingan expression vector of claim
 4. 6. The host cell of claim 5, whereinsaid host cell is a mammalian cell.
 7. The host cell of claim 5, whereinsaid host cell is a bacterial cell.
 8. The host cell of claim 5, whereinsaid host cell is a yeast cell.
 9. The host cell of claim 5, whereinsaid host cell is an insect cell.
 10. The host cell of claim 5, whereinsaid host cell is a plant cell.
 11. A polypeptide comprising an aminoacid sequence encoded by a cDNA derived nucleic acid sequence thatencodes an amino acid sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8 or SEQ ID NO:
 10. 12. A method of restoring photosensitivity to aretina of a subject suffering from vision loss or blindness, said methodcomprising: (a) delivering to the retina of said subject an expressionvector comprising a polynucleotide that encodes an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 whichencodes a rhodopsin domain of a channelrhodopsin expressible in aretinal neuron; and (b) expressing said vector in said retinal neuron,wherein the expressed rhodopsin renders said retinal neuronphotosensitive, thereby restoring photosensitivity to said retina or aportion thereof.
 13. The method of claim 12 wherein, said methodcomprises: (a) delivering to the retina of said subject an expressionvector that encodes a type 1 rhodopsin domain; said vector comprising anopen reading frame encoding the type 1 rhodopsin domain of a red-shiftedchannelrhodopsin selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10 andoperatively linked to a promoter sequence; and (b) expressing saidvector in said retinal neuron, wherein the expressed rhodopsin renderssaid retinal neuron photosensitive, thereby restoring photosensitivityto said retina or a portion thereof.